Synthetic apolipoprotein e mimicking polypeptides and methods of use

ABSTRACT

The present invention provides novel synthetic apolipoprotein E (ApoE)-mimicking peptides wherein the receptor binding domain of apolipoprotein E is covalently linked to 18A, the well characterized lipid-associating model class A amphipathic helical peptide, or a modified version thereof. Such peptides enhance low density lipoprotein (LDL) and very low density lipoprotein (VLDL) binding to and degradation by fibroblast or HepG2 cells. Also provided are possible applications of the synthetic peptides in lowering human plasma LDL/VLDL cholesterol levels, thus inhibiting atherosclerosis. The present invention also relates to synthetic peptides that can improve HDL function and/or exert anti-inflammatory properties.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 12/675,073, which is national phase application ofInternational Application No. PCT/US2008/074485, filed on Aug. 27, 2008,which claims priority to U.S. Provisional Patent Application No.60/968,355, filed on Aug. 27, 2007, all of which are hereby incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology and proteinbiology including polypeptides and polypeptide mimics. This applicationalso relates to the field of cholesterol metabolism, catabolism, and thetreatment and management of cholesterol associated disorders. Thepresent invention also relates generally to the field of cardiovascularmedicine. More specifically, the present invention relates to syntheticpeptides that can rapidly lower plasma cholesterol through enhanced LDLand VLDL uptake and degradation by cells. The present invention alsorelates to synthetic peptides that can improve HDL function and/or exertanti-inflammatory properties.

BACKGROUND OF THE INVENTION

Epidemiological studies indicate that increased plasma cholesterollevels increase the risk for atherosclerosis. Five completed majortrials have provided conclusive evidence of a benefit from treatmentaimed primarily at reducing low-density lipoprotein (LDL)-cholesterol(Illingworth R D., et al. Current Opini. Lipidol. 1999, 10:383-386).Among other lipoprotein risk factors is familial dysbetalipoproteinemia,which results in the accumulation of remnant atherogenic lipoproteinsderived from the catabolism of chylomicron and VLDL (Kwiterovich, P. O.,Jr. Am. J. Cardiol. 1998, 82:3 U-7U). It has been shown that a 1%decrease in the plasma cholesterol level decreases the risk of coronaryartery disease by 2% (Deedwania, P. C. Med. Clin. North Am. 1995,79:973-998). The focus of angiographic trials has been on LDL reductionand these studies have demonstrated that decreases in LDL-cholesterol ofmore than 30% to 35% are associated with lower rates of coronary events(Watts, G. W., et al. Atherosclerosis 1998, 414:17-30). There is alsogrowing evidence that triglyceride-rich lipoproteins may adverselyaffect endothelial function and increase oxidative stress by promotingthe production of small, dense LDL and by reducing high-densitylipoprotein (HDL) levels (Marais, D., Curr. Opin. Lipidol. 2000,11:597-602).

Apolipoprotein E (apo E) plays an important role in the metabolism oftriglyceride-rich lipoproteins, such as very low density lipoprotein(VLDL) and chylomicrons. Apolipoprotein E mediates the high affinitybinding of apo E-containing lipoproteins to the low density lipoprotein(LDL) receptor (apo B, E receptor) and the members of its gene family,including LDL receptor related protein (LRP), very low densitylipoprotein receptor (VLDLR) and the apoE2 receptor (apoE2R) (Mahley, R.W., (1988) Science 240, 622-630). The putative and complex role of apo Ein atherosclerosis has been emphasized by several observations: (i) micethat overexpress human apo E have lower levels of total plasmacholesterol levels (Shimono, H. N., et al., (1992) Eur. J. Clin. Invest.90, 2084-2991), (ii) intravenous injection of human apo E intocholesterol-fed rabbits protects these animals from atherosclerosis(Yamada, et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 665-669), and(iii) loss of the apo E gene in mice produces spontaneousatherosclerosis (Zhang, S. H., et al., (1992) Science 258, 468-471)which is ameliorated when macrophage-specific apo E expression isinitiated in apo E-deficient mice (Spangenberg, J., et al., (1997)Biochem. Biophys. Acta 1349, 109-121).

Apolipoprotein E is a protein that binds lipid and has two major domains(Mahley, R. W., et al. J. Lipid Res. 1999, 40:622-630). The 22 kDa aminoterminal domain has been shown by X-ray crystallographic studies to be a4-helix bundle (Wilson, C., et al. Science 1991; 252:1817-1822) and tocontain a positively-charged receptor binding domain. For this region tomediate very low-density lipoprotein (VLDL) binding to its receptors,the apolipoprotein must associate with the lipoprotein surface; this isenabled by the C-terminal amphipathic helical region. If the 4-helixbundle that contains the positively charged receptor-binding domain doesnot open up on the lipoprotein surface, then the VLDL is defective inbinding to receptors. Thus, the positively charged arginine (Arg)-richcluster domain of the Apo E and the C-terminal amphipathic helicaldomain, are both required for the enhanced uptake of atherogenic ApoE-containing lipoproteins.

Apo E is secreted as a 299 amino acid residue protein with a molecularweight of 34,200. Based on thrombin cleavage of apo E into twofragments, a two-domain hypothesis was initially suggested to explainthe fact that the C-terminal region of apo E (192-299) is essential forits binding to hypertriglyceridemic VLDL and the N-terminal 22 kDadomain (1-191), binds to the LDL-R (Bradley, W. A., et al., (1986) J.Lipid Res. 27, 40-48). Additional physical-chemical characterization ofthe protein and its mutants have extended this concept and have shownthat the region 192-211 binds to phospholipid while the amino terminaldomain (1-191) is a globular structure that contains the LDL receptorbinding domain in the 4-helix bundle (Wilson, C., et al., (1991) Science252, 1817-1822). Studies with synthetic peptides (Sparrow et al.) andmonoclonal antibodies pinpointed the LDL receptor binding domain of apoE between residues 129-169, a domain enriched in positively chargedamino acids, Arg and Lys (Rall, S. C., Jr., et al., (1982) PNAS USA 79,4696-4700; Lalazar, A., et al., (1988) J. Biol. Chem. 263, 3542-2545;Dyer, C. A., et al., (1991) J. Biol. Chem. 296, 22803-22806; and Dyer,C. A., et al., (1991) J. Biol. Chem. 266, 15009-15015).

Further studies with synthetic peptides were used to characterize thestructural features of the domain of apo E that mediates its interactionwith the LDL receptor (Dyer, C. A., et al., (1991) J. Biol. Chem. 296,22803-22806; Dyer, C. A., et al., (1991) J. Biol. Chem. 266,15009-15015; and Dyer, C. A., et al., (1995) J. Lipid Res. 36, 80-8).Residues 141-155 of apo E, although containing the positively chargedresidues, did not compete for binding of LDL in a human skin fibroblastassay, but did so only as tandem covalent repeats [i.e. (141-155)₂].N-acetylation of the (141-155)₂ peptide, on the other hand, enhanced LDLbinding to fibroblasts (Nicoulin, I. R., et al., (1998) J. Clin Invest.101, 223-234). The N-acetylated (141-155)₂ analog selectively associatedwith cholesterol-rich lipoproteins and mediated their acute clearance invivo (Nicoulin, I. R., et al., (1998) J. Clin Invest. 101, 223-234).Furthermore, these studies indicated that the prerequisite for receptorbinding is that the peptides be helical (Dyer, C. A., et al., (1995) J.Lipid Res. 36, 80-88). Enhanced LDL uptake and degradation were alsoobserved (Mims, M. P., et al., (1994) J. Biol. Chem. 269, 20539-20647)using synthetic peptides modified to increase lipid association byN,N-distearyl derivation of glycine at the N-terminus of the native129-169 sequence of Apo E (Mims, M. P., et al., (1994) J. Biol. Chem.269, 20539-20647). Although LDL binding is mediated by the cationicsequence 141-155 of human Apo E, Braddock et al. (Braddock. D. T., etal., (1996) Biochemistry 35, 13975-13984) have shown that model peptidesof the highly conserved anionic domain (41-60 of human Apo E) alsomodulate the binding and internalization of LDL to cell surfacereceptors. However, these peptides do not enhance LDL degradation.

Chylomicron is a lipoprotein found in blood plasma, which carries lipidsfrom the intestines into other body tissues and is made up of a drop oftriacylglycerols surrounded by a protein-phospholipid coating.Chylomicron remnants are taken up by the liver (Havel, R. J., 1985,Arteriosclerosis. 5:569-580) after sequestration in the space of Disse,which is enhanced in the presence of Apo E (Kwiterovich, P. O., Jr.,1998; Deedwania, P. C., 1995; and Watts, G. W., et al., 1998). Apo E isthe major mediator of hepatic remnant lipoprotein uptake by the LDLreceptor or LRP. Lipolysis of normal VLDL Sf (subfraction) of more than60 permit binding of the lipolytic remnant to the LDL receptor(Catapano, A. L. et al., 1979, J. Biol. Chem. 254:1007-1009; Schonfield,G., et al. 1979. J. Clin. Invest. 64:1288-1297). Lipoprotein lipase(LpL) may facilitate uptake through localization of Apo B-containinglipoproteins to membrane heparan sulphate proteoglycan (HSPG)(Eisenberg, et al. 1992. J. Clin. Invest. 90:2013-2021; Hussain, M., etal., J. Biol. Chem. 2000, 275:29324-29330) and/or through binding to theLDL-receptor-related protein (LRP) (Beisiegel, U., et al., 1989, Nature341:162-164). Cell-surface HSPG may also function as a receptor and hasvariable binding affinities for specific isoforms of Apo E. Inparticular, Apo E is synthesized by the liver and also bymonocyte/macrophages, where it exerts its effect on cholesterolhomeostasis. In vivo evidence for the local effect of lack of Apo Ecomes from the observations of Linton and Fazio, who showed acceleratedatherosclerosis in C57BL/6 mice transplanted with bone marrow from ApoE-deficient mice (Linton, M. F. and Fazio, S. Curr. Openi. Lipidol.1999, 10:97-105). Apo E-dependent LDL cholesteryl ester uptake pathwayhas been demonstrated in murine adrenocortical cells (Swarnakar, S., etal. J. Biol. Chem. 2001, 276:21121-21126). This appears to involvechondroitin sulphate proteoglycan (CSPG) and a 2-macroglobulin receptor.

It has been shown that the receptor-binding domain of Apo E, rich in Argresidues (141-150), covalently linked to a synthetic class Aamphipathic-helical domain, enhances the hepatic atherogenic lipoproteinuptake (Datta, G., et al. Biochemistry 2000, 30:213-220). Recent studiesindicate that a potential anti-atherogenic action of Apo E is that itstimulates endothelial production of heparan sulfate (HS) (Paka, L., etal. J. Biol. Chem. 1999, 274:4816-4823). Lipoproteins are complexes ofone or more lipids bound to one or more proteins and transportwater-insoluble fats in the blood. Cholesterol is carried through thebloodstream by lipoproteins. There are no agents available which reducecholesterol via the binding mechanisms of lipoproteins. There is a needfor more effective agents that are capable of reducing cholesterol in asubject so as to reduce diseases and conditions which are associatedwith increased cholesterol.

U.S. Pat. No. 6,506,880 denotes the first effort to synthesizeapolipoprotein E-mimicking peptides based on the hypothesis that sincelipid binding is essential for surface localization of the peptide onlipoproteins and for the receptor binding domain of apo E to beappropriately accessible to bind to the LDL receptor, joining awell-characterized, lipid-associating peptide such as the model class Aamphipathic helix, 18A, to the 141-150 peptide sequence of apo E shouldbe sufficient to confer biological activity. It was found that thepeptides enhanced LDL/VLDL binding to a cell, increased LDL/VLDLdegradation by a cell, lowered LDL/VLDL cholesterol in an in-needindividual with atherosclerosis.

The present invention provides novel synthetic apolipoprotein E(ApoE)-mimicking peptides wherein the receptor binding domain ofapolipoprotein E is covalently linked to 18A, the well characterizedlipid-associating model class A amphipathic helical peptide as well aspossible applications of the synthetic peptides in lowering human plasmaLDL/VLDL cholesterol levels, thus inhibiting atherosclerosis. Thepresent invention also provides possible applications of the syntheticpeptides to improve HDL function and/or exert anti-inflammatoryproperties.

SUMMARY OF THE INVENTION

The present invention provides polypeptides, compositions and methods ofuse of said polypeptides and compositions. Disclosed herein aresynthetic apolipoprotein E-mimicking peptides. For example, disclosed isa synthetic apolipoprotein E-mimicking peptide, consisting of: areceptor binding domain of apolipoprotein E comprising the amino acidsequence of SEQ ID NO: 15; and a lipid-associating peptide, wherein saidreceptor binding domain is covalently linked to said lipid-associatingpeptide. The lipid-associating peptide of the disclosed syntheticapolipoprotein E-mimicking peptides can be model class A amphipathichelical peptide 18A. For example, the lipid-associating peptide cancomprise the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of: a receptor binding domain of apolipoprotein E comprisingthe amino acid sequence of SEQ ID NO: 15; and a lipid-associatingpeptide, wherein said receptor binding domain is covalently linked tosaid lipid-associating peptide, wherein said synthetic peptide isprotected using acetyl and amide groups at the N- and C-terminus,respectively. The disclosed synthetic apolipoprotein E-mimickingpeptides can also be N-terminally protected with an acetyl group. Thedisclosed synthetic apolipoprotein E-mimicking peptides can also beC-terminally protected with an amide group.

Also disclosed herein are synthetic apolipoprotein E-mimicking peptides,comprising: a lipid binding domain of apolipoprotein E comprising theamino acid sequence of SEQ ID NO: 17; and a receptor binding domainpeptide, wherein said lipid binding domain is covalently linked to saidreceptor binding domain peptide. The receptor binding domain peptide ofsuch synthetic apolipoprotein E-mimicking peptides can be a humanreceptor binding domain peptide of ApoE. For example, receptor bindingdomain peptide of these synthetic apolipoprotein E-mimicking peptidescan comprise the amino acid sequence of SEQ ID NOs: 1 or 15. Thereceptor binding domain peptide of these synthetic apolipoproteinE-mimicking peptides can also comprise the amino acid sequence of SEQ IDNOs: 2, 3, 5, 6, 7, 8, 9, or 10.

Also disclosed herein are synthetic apolipoprotein E-mimicking peptides,comprising: a lipid binding domain of apolipoprotein E comprising theamino acid sequence of SEQ ID NO: 17; and a receptor binding domainpeptide, wherein said lipid binding domain is covalently linked to saidreceptor binding domain peptide, wherein said synthetic peptide isprotected using acetyl and amide groups at the N- and C-terminal ends,respectively. Also disclosed are synthetic apolipoprotein E-mimickingpeptides, wherein the synthetic apolipoprotein E-mimicking peptides canbe from a species selected from the group consisting of human, mouse,rabbit, monkey, rat, bovine, pig and dog.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of a combination of the disclosed receptor binding domains ofapolipoprotein E and the disclosed lipid-associating peptides, whereinsaid receptor binding domain is covalently linked to saidlipid-associating peptide in a reversed orientation. Also disclosed aresynthetic apolipoprotein E-mimicking peptides, consisting of acombination of the disclosed receptor binding domains of apolipoproteinE and the disclosed lipid-associating peptides, wherein said receptorbinding domain is covalently linked to said lipid-associating peptide ina domain switched orientation.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide, wherein thereceptor binding domain of apolipoprotein E is scrambled. Also disclosedare synthetic apolipoprotein E-mimicking peptides, consisting of: areceptor binding domain of apolipoprotein E and a lipid-associatingpeptide, wherein said receptor binding domain is covalently linked tosaid lipid-associating peptide, wherein the lipid-associating peptide ofapolipoprotein E is scrambled. Also disclosed are syntheticapolipoprotein E-mimicking peptides, consisting of: a receptor bindingdomain of apolipoprotein E and a lipid-associating peptide, whereinreceptor binding domain is covalently linked to said lipid-associatingpeptide, wherein both the receptor binding domain of apolipoprotein Eand the lipid-associating peptide of apolipoprotein E are scrambled.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide, wherein receptor binding domain is covalentlylinked to said lipid-associating peptide, wherein either the receptorbinding domain of apolipoprotein E or the lipid-associating peptide ofapolipoprotein E, or both are scrambled and the peptide isreverse-oriented. Also disclosed are pharmaceutical compositionscomprising the disclosed synthetic apolipoprotein E-mimicking peptidesand a pharmaceutically acceptable carrier. Also disclosed are isolatednucleic acids encoding the disclosed synthetic apolipoproteinE-mimicking peptides. For example, disclosed are isolated nucleic acidencoding the disclosed synthetic apolipoprotein E-mimicking peptides,wherein the nucleic acid comprises DNA, RNA and/or cDNA.

Also disclosed are vectors comprising isolated nucleic acids encodingthe disclosed synthetic apolipoprotein E-mimicking peptides. Alsodisclosed are host cells comprising isolated nucleic acids encoding thedisclosed synthetic apolipoprotein E-mimicking peptides. For example,disclosed are eukaryotic host cells and a prokaryotic host cellscomprising isolated nucleic acids encoding the disclosed syntheticapolipoprotein E-mimicking peptides. Also disclosed are recombinantcells comprising isolated nucleic acids encoding the disclosed syntheticapolipoprotein E-mimicking peptides.

Also disclosed are recombinant cells producing the disclosed syntheticapolipoprotein E-mimicking peptides. Also disclosed are antibodies thatbind the disclosed synthetic apolipoprotein E-mimicking peptides. Alsodisclosed are transgenic, non-human subjects comprising isolated nucleicacids encoding the disclosed synthetic apolipoprotein E-mimickingpeptides. For example, disclosed a transgenic animal and plantscomprising isolated nucleic acids encoding the disclosed syntheticapolipoprotein E-mimicking peptides.

Also disclosed are transgenic, non-human subjects expressing thedisclosed synthetic apolipoprotein E-mimicking peptides. Also disclosedare methods for enhancing LDL binding to a cell, the method comprisingcontacting the cell with the disclosed synthetic apolipoproteinE-mimicking peptides. Also disclosed are methods comprisingadministering the disclosed synthetic apolipoprotein E-mimickingpeptides to a subject, whereby plasma LDL, plasma VLDL, or both, areaffected.

Also disclosed are methods comprising administering the disclosedsynthetic apolipoprotein E-mimicking peptides to a subject, wherebyplasma LDL, plasma VLDL, or both, are affected, wherein the syntheticapolipoprotein E-mimicking peptide is administered as a compositioncomprising the synthetic apolipoprotein E-mimicking peptide and apharmaceutically acceptable carrier. Also disclosed are methodscomprising administering the disclosed synthetic apolipoproteinE-mimicking peptides to a subject, whereby plasma LDL, plasma VLDL, orboth, are affected, wherein binding of LDL to a cell of the subject isenhanced, degradation of LDL by a cell of the subject is increased, LDLcholesterol in the subject is lowered, binding of VLDL to a cell of thesubject is enhanced, degradation of VLDL by a cell of the subject isincreased, VLDL cholesterol in the subject is lowered, and/or totalplasma concentration of cholesterol in the subject is lowered.

Also disclosed are methods comprising administering the disclosedsynthetic apolipoprotein E-mimicking peptides to a subject, wherebyplasma LDL, plasma VLDL, or both, are affected, wherein said syntheticapolipoprotein E-mimicking peptide is administered in an amount of about0.01 mg/kg to about 5 mg/kg. Also disclosed are methods comprisingadministering the disclosed synthetic apolipoprotein E-mimickingpeptides to a subject, whereby plasma LDL, plasma VLDL, or both, areaffected, wherein the subject has coronary artery disease, rheumatoidarthritis, and/or systemic lupus.

Also disclosed are methods for treating a subject with a “LipidDisorder”, the method comprising administering to the subject aneffective amount of the disclosed synthetic apolipoprotein E-mimickingpeptides, or a composition thereof. Also disclosed are methods fortreating a subject with a “Lipid Disorder”, the method comprisingadministering to the subject an effective amount of the disclosedsynthetic apolipoprotein E-mimicking peptides, or a composition thereof,wherein the synthetic apolipoprotein E-mimicking peptide is administeredas a composition comprising the synthetic apolipoprotein E-mimickingpeptide and a pharmaceutically acceptable carrier. Also disclosed aremethods for treating a subject with a “Lipid Disorder”, the methodcomprising administering to the subject an effective amount of thedisclosed synthetic apolipoprotein E-mimicking peptides, or acomposition thereof, wherein binding of LDL to a cell of the subject isenhanced, degradation of LDL by a cell of the subject is increased, LDLcholesterol in the subject is lowered, binding of VLDL to a cell of thesubject is enhanced, degradation of VLDL by a cell of the subject isincreased, VLDL cholesterol in the subject is lowered, and/or totalplasma concentration of cholesterol in the subject is lowered.

Also disclosed are methods for treating a subject with a “LipidDisorder”, the method comprising administering to the subject aneffective amount of the disclosed synthetic apolipoprotein E-mimickingpeptides, or a composition thereof, wherein said syntheticapolipoprotein E-mimicking peptide is administered in an amount of about0.01 mg/kg to about 5 mg/kg. Also disclosed are methods for treating asubject with a “Lipid Disorder”, the method comprising administering tothe subject an effective amount of the disclosed syntheticapolipoprotein E-mimicking peptides, or a composition thereof, whereinthe subject has coronary artery disease, rheumatoid arthritis, and/orsystemic lupus.

Also disclosed are methods for reducing serum cholesterol in a subject,the method comprising administering to the subject an effective amountof the disclosed synthetic apolipoprotein E-mimicking peptides, or acomposition thereof. Also disclosed are methods for reducing serumcholesterol in a subject, the method comprising administering to thesubject an effective amount of the disclosed synthetic apolipoproteinE-mimicking peptides, or a composition thereof, wherein the syntheticapolipoprotein E-mimicking peptide is administered as a compositioncomprising the synthetic apolipoprotein E-mimicking peptide and apharmaceutically acceptable carrier.

Also disclosed are methods for reducing serum cholesterol in a subject,the method comprising administering to the subject an effective amountof the disclosed synthetic apolipoprotein E-mimicking peptides, or acomposition thereof, wherein binding of LDL to a cell of the subject isenhanced, degradation of LDL by a cell of the subject is increased, LDLcholesterol in the subject is lowered, binding of VLDL to a cell of thesubject is enhanced, degradation of VLDL by a cell of the subject isincreased, VLDL cholesterol in the subject is lowered, and/or totalplasma concentration of cholesterol in the subject is lowered.

Also disclosed are methods for reducing serum cholesterol in a subject,the method comprising administering to the subject an effective amountof the disclosed synthetic apolipoprotein E-mimicking peptides, or acomposition thereof, wherein said synthetic apolipoprotein E-mimickingpeptide is administered in an amount of about 0.01 mg/kg to about 5mg/kg. Also disclosed are methods for reducing serum cholesterol in asubject, the method comprising administering to the subject an effectiveamount of the disclosed synthetic apolipoprotein E-mimicking peptides,or a composition thereof, wherein the subject has coronary arterydisease, rheumatoid arthritis, and/or systemic lupus.

Also disclosed are methods for enhancing HDL function, the methodscomprising contacting the cell with the disclosed syntheticapolipoprotein E-mimicking peptides. Also disclosed are methods fordecreasing inflammation, the methods comprising contacting the cell withthe disclosed synthetic apolipoprotein E-mimicking peptides, wherein thepeptides remove the lipid hydro-peroxides from the plasma by increasingparaoxanase.

Also disclosed are methods for increasing plasma paraoxonase (PON-1)activity, the methods comprising contacting the cell with the disclosedsynthetic apolipoprotein E-mimicking peptides. Also disclosed aremethods for inhibiting atherogenesis, the methods comprising contactingthe cell with the disclosed synthetic apolipoprotein E-mimickingpeptides. Also disclosed are methods for inhibiting atherogenesis, themethods comprising contacting the cell with the disclosed syntheticapolipoprotein E-mimicking peptides, wherein plasma cholesterol levelsare decreased and HDL functions are increased.

Also disclosed are methods for removing atherogenic lipoproteins fromvessel walls, the methods comprising contacting the cell with thedisclosed synthetic apolipoprotein E-mimicking peptides. Also disclosedare methods for decreasing the atherogenicity of LDL, the methodscomprising contacting the cell with the disclosed syntheticapolipoprotein E-mimicking peptides

Also disclosed are methods comprising administering the disclosedsynthetic apolipoprotein E-mimicking peptides to a subject, wherebyplasma HDL is affected. Also disclosed are methods comprisingadministering the disclosed synthetic apolipoprotein E-mimickingpeptides to a subject, whereby plasma HDL is affected, wherein thesynthetic apolipoprotein E-mimicking peptide is administered as acomposition comprising the synthetic apolipoprotein E-mimicking peptideand a pharmaceutically acceptable carrier. Also disclosed are methodscomprising administering the disclosed synthetic apolipoproteinE-mimicking peptides to a subject, whereby plasma HDL is affected,wherein PON activity is increased, lipid hydroperoxides are cleared,atherogenic lipoproteins levels are reduced in the plasma, endothelialfunction is improved, and/or atherogenic lipoproteins are removed fromthe vessel wall.

Also disclosed are methods comprising administering the disclosedsynthetic apolipoprotein E-mimicking peptides to a subject, wherebyplasma HDL is affected, wherein the subject has Inflammatory BowelDisease (IBD), systemic lupus erythematosus, Hashimoto's disease,rheumatoid arthritis, graft-versus-host disease, Sjögren's syndrome,pernicious anemia, Addison disease, Alzheimer's disease, scleroderma,Goodpasture's syndrome, ulcerative colitis, Crohn's disease, autoimmunehemolytic anemia, sterility, myasthenia gravis, multiple sclerosis,Basedow's disease, thrombopenia purpura, allergy; asthma, atopicdisease, cardiomyopathy, glomerular nephritis, hypoplastic anemia,metabolic syndrome X Synthetic Apolipoprotein E Mimicking Polypeptidesand Methods of Use, peripheral vascular disease, chronic obstructivepulmonary disease (COPD), emphysema, asthma, idiopathic pulmonaryfibrosis, pulmonary fibrosis, adult respiratory distress syndrome,osteoporosis, Paget's disease, coronary calcification, polyarteritisnodosa, polymyalgia rheumatica, Wegener's granulomatosis, centralnervous system vasculitis (CNSV), Sjogren's syndrome, scleroderma,polymyositis, AIDS inflammatory response, influenza, avian flu, viralpneumonia, endotoxic shock syndrome, sepsis, sepsis syndrome,trauma/wound, corneal ulcer, chronic/non-healing wound, reperfusioninjury (prevent and/or treat), ischemic reperfusion injury (preventand/or treat), spinal cord injuries (mitigating effects), cancers,myeloma/multiple myeloma, ovarian cancer, breast cancer, colon cancer,bone cancer, osteoarthritis, allergic rhinitis, cachexia, Alzheimer'sdisease, implanted prosthesis, biofilm formation, dermatitis, acute andchronic, eczema, psoriasis, contact dermatitis, erectile dysfunction,macular degeneration, nephropathy, neuropathy, Parkinson's Disease,peripheral vascular disease, and meningitis, cognition and rejectionafter organ transplantation.

Also disclosed are methods for treating a subject with an “InflammatoryDisorder”, the method comprising administering to the subject aneffective amount of the disclosed synthetic apolipoprotein E-mimickingpeptides, or a composition thereof. Also disclosed are methods fortreating a subject with an “Inflammatory Disorder”, the methodscomprising administering to the subject an effective amount of thedisclosed synthetic apolipoprotein E-mimicking peptides, or acomposition thereof, wherein the synthetic apolipoprotein E-mimickingpeptide is administered as a composition comprising the syntheticapolipoprotein E-mimicking peptide and a pharmaceutically acceptablecarrier. Also disclosed are synthetic apolipoprotein E-mimickingpeptides consisting of a receptor binding domain of apolipoprotein E anda lipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide in a domain switchedorientation.

Also disclosed are synthetic apolipoprotein E-mimicking peptidesconsisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide, wherein thereceptor binding domain of apolipoprotein E is in a reversed orientationAlso disclosed are synthetic apolipoprotein E-mimicking peptidesconsisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide, wherein thelipid-associating peptide is in a reversed orientation.

Also disclosed are synthetic apolipoprotein E-mimicking peptidesconsisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide, wherein both thereceptor binding domain of apolipoprotein E and the lipid-associatingpeptide are in a reversed orientation. Also disclosed are syntheticapolipoprotein E-mimicking peptides consisting of a receptor bindingdomain of apolipoprotein E. Also disclosed are synthetic apolipoproteinE-mimicking peptides consisting of a receptor binding domain ofapolipoprotein E wherein the receptor binding domain is modified oraltered.

Also disclosed are synthetic apolipoprotein E-mimicking peptidesconsisting of a receptor binding domain of apolipoprotein E wherein thereceptor binding domain is mutated, scrambeled, and/or reverse-oriented.Also disclosed are synthetic apolipoprotein E-mimicking peptidesconsisting of a lipid-associating peptide. Also disclosed are syntheticapolipoprotein E-mimicking peptides consisting of a lipid-associatingpeptide wherein the lipid-associating peptide is modified or altered.Also disclosed are synthetic apolipoprotein E-mimicking peptidesconsisting of a lipid-associating peptide wherein the lipid-associatingpeptide is mutated, scrambeled, and/or reverse-oriented.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention. These are non-limiting examples.

FIG. 1 shows a helical net representation of the difference betweenhE-18A and a scrambled form of hE-18A. As seen on the left side of thefigure, the “α-amphipathic helix” has 3.6 amino acid residues per turnof the helix, whereas the “π-helix” has 4.4 amino acid residues perturn. The helical net does not show segregation of faces, thus theamphipathic helix nature is lost. Helical net is 2-dimensionalrepresentation of the helix cylinder when it is cut horizontally t thecenter of the cylinder and laid flat.

FIG. 2 shows the effect of Ac-hE18A-NH₂ (i.v.-administration) for 4weeks in apo E knock-out mice (16 weeks) on lesion formation. Extent oflesion is analyzed by en face preparation and staining with Oil Red O.

FIG. 3 shows the long-term effect of one-time administration ofAc-hE-18A-NH₂ (n=9 in each group). An initial reduction in plasmacholesterol is followed by cholesterol levels coming back to originalvalues at 24 h. A significant decrease was observed at 4-days and wasmaintained for 8 days.

FIG. 4 shows HepG2 cells that were incubated with ¹²⁵I-Ac-hE18A-NH₂ for5 minutes and 60 minutes and the peptide releasable by heparin andheparinase/heparitinase (H/H) determined. The percent of peptidereleased by H/H after 60 min incubation is more than that observed at 5min while there is less peptide in the cells.

FIG. 5 shows Apo A-I secretion of HepG2 cells treated with peptides:Ac-hE-4F—NH₂ (I), Ac-hE-18A-NH₂(III), and 4F(II), at 24, 48 and 72 htime points: Cells grown to confluency, and treated with peptide (50μg/ml), in media without FBS. Media containing peptide was removed after1^(st) O/N incubation, and replaced with media (without peptide) w/oFBS. After the 2^(nd) O/N incubation, media was removed and replacedwith media w/o FBS, and incubated for the third and final night. Agarosegels were run for each time point. Western blots were performed forhuman Apo A-I, to determine the distribution of preβ-HDL particles.C=control cells without peptide. These results show that the peptide haseffect for a longer period since it is internalized and re-released.

FIG. 6 shows a Western blot for VCAM expression. HUVECs were challengedwith the peptide alone, peptide+LPS and LPS. LPS induces expression ofVCAM-1 (lane 3). Peptide by itself (lane 1) does not show any adverseeffect, while it inhibits the expression of VCAM-1 induced by LPS bymore than 80% (lane 2). These results indicate that the peptide has ananti-inflammatory effect.

FIG. 7 shows treatment of THP-1 derived macrophages with Ac-hE-18A-NH₂enhances the synthesis of apo E. Cells were metabolically labeled with³⁵S-methionine, treated with the peptide (25 μg/10⁶ cells) for 5 h andthe medium was subjected to SDS-PAGE. Bands were developed byautoradiography and quantitated by densitiometry. These results indicatethat the peptide can stimulate apo E synthesis and the chronic effect ofthe peptide on cholesterol reducing ability and anti-inflammatoryability is partly due to its ability to promote apo E synthesis.

FIG. 8 shows the effect of oral feeding of 18L-2Y and R18L-2Y (1mg/mouse) for 6 weeks in female apo E ko mice. 4 week old female apo Eknock-out mice that were fed with peptides 18L-2Y and R18L-2Y for 6weeks. The peptides were mixed in normal chow (1 mg/4 g chow) and fed adlibitum. At the end of 6 weeks, the animals were euthanized and theatherosclerotic lesion area was stained with Oil Red O and quantified.n=20 for control (solid black) and 18L-2Y treated group (light grey) andn=23 in R18L-2Y treated group (dark grey). †, p<0.01 vs control (dark)and ‡, p<0.01 vs 18L-2Y.

FIG. 9 shows the effect of Ac-hE18A-NH₂ on mRNA levels in THP-1 derivedmacrophages.

FIG. 10 shows a schematic representation of the proatherogenic effectswithout administration of one of the disclosed peptides and that one ofthe disclosed peptides can correct this by an antiinflamatory mechanism.

FIG. 11 shows plasma cholesterol levels over time in rabbitsadministered with Ac-hE18A-NH₂. Administration of Ac-hE18A-NH₂ to highfat diet administered rabbits with initial cholesterol values in therange of 600 mg/dl (1 week on 1% cholesterol diet). Peptide (5 mg/kg)was iv-administered two times as shown in the figure (n=4). At the endof 14 days (21 days after the initiation of atherogenic diet), whileplasma cholesterol levels in the control rabbits were in the range of2000 mg/dl (n=4), the peptide administered rabbits showed cholesterolvalues in the range of 1000 mg/dl. A 50% decrease in plasma cholesterolwas observed after administration of the peptide.

FIG. 12 shows turn over experiments in NZW rabbits fed 1% diet showsinitial decreases cholesterol (and the disappearance of peptide) fromplasma. Despite the loss of peptide from the plasma, effect of thepeptide lasts for 14 days.

FIG. 13 shows aortal rings in control, atherogenic diet administered anddiet-administered with peptide i.v. administered rabbits were studiedfor endothelial function. While the diet administered rabbit aortalrings did not respond to acetyl choline, aortae from rabbits on high fatdiet and peptide-administered rabbits showed dose-dependent relaxationto acetyl choline, almost similar to aortae from normaldiet-administered rabbits.

FIG. 14 shows that class A peptides inhibit 18L-induced lysis: Molecularbasis for this inhibition is the opposite cross-sectional shape of thesemolecules. If K in 18L is replaced by R, lytic activity is reduced tominimum, due to the change in the cross-sectional shape in the peptideR18L to trapezoidal.

FIG. 15 shows a rational design of R18L-2Y to reduce lytic propertiesand enhance uptake of atherogenic lipoproteins.

FIG. 16 shows the rationale for selecting R18L-2Y for further studiesEffect of 18L-2Y and R18L-2Y on plasma cholesterol in E−/− mice(Dose—100 μg i.v.).

FIG. 17 shows oral administration of R18L-2Y decreases plasmacholesterol in apo E null mice. Peptide, 1 mg/4 g of chow (per animalper day) (apo E−/− mice) lowers plasma cholesterol (1 mg/mouse/day) for30 days. (n=5 in each group).

FIG. 18 shows the effect of peptide R18L-2Y (1 mg/4 g of chow)administration on plasma cholesterol levels.

FIG. 19 shows peptide-Ac-hE-18A-NH₂-mediated improvement of HDLfunction.

FIG. 20 shows the timeline of hE-4F, hE-Sc2F and L-4F administration toZDF rats. Peptides were administered to the rats intravenously at aconcentration of 5 mg/kg.

FIG. 21 shows a helical wheel representation of the peptide sequence 4Fas a scrambled 4F peptide.

FIG. 22 shows the effect of three peptides on plasma cholesterol in apoE null mice at two different time points (5 minutes and 2 hours). Thepeptides represented are Ac-hE-18A-NH₂, Ac-hE4F—NH₂, and Ac-hE-Sc2F—NH₂.Peptides Ac-hE-18A-NH₂, Ac-hE-4F—NH₂ and Ac-hE-Sc 18A were administered(i.v.) to apo E null mice (n=4) and plasma cholesterol values weredetermined at before administration (0 min), 5 min and 2 h afteradministration. While Ac-hE-18A-NH₂ and Ac-hE-4F—NH₂ show a higherreduction in plasma cholesterol levels at 2 h time point, peptideAc-hE-Sc18A-NH₂ did not show much difference.

FIG. 23 shows Sc-hE-18A plotted as an α-helix or a π-helix. In thesequence Sc-hE-18A (LRLLRKLKRR-DWLKAFYDKVEKLKEAF), the hE-portion isscrambled. When this is scrambled, the sequence, when folded as alphahelix (3.6 residues/turn), the resulting alpha helix is not anamphipathic helix since there is no segregation of two (polar andnonpolar) faces. However, if it is folded as a pi-helix (4.4residues/turn), the resulting structure also does not show segregationof polar and nonpolar faces.

FIG. 24 shows a helical net representation of hE-Sc-18A. In this helicalnet program (that is, peptide sequence is folded a alpha or pi helix andspread on a plane) the alpha helix does not show segregation of poparand nonpolar faces whereas the pi-helix shows a clear nonpolar face atthe center (black circles) and the polar residues blue and red circlesappear at the edge. Peptides may associate with lipid as a pi-helix.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications and publications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretiesinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

It is to be understood that this invention is not limited to specificsynthetic methods, or to specific recombinant biotechnology methodsunless otherwise specified, or to particular reagents unless otherwisespecified, to specific pharmaceutical carriers, or to particularpharmaceutical formulations or administration regimens, as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

A. DEFINITIONS AND NOMENCLATURE

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” can include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a compound”includes mixtures of compounds, reference to “a pharmaceutical carrier”includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 20%. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

As used herein, the term “amino acid sequence” refers to a list ofabbreviations, letters, characters or words representing amino acidresidues. The amino acid abbreviations used herein are conventional oneletter codes for the amino acids and are expressed as follows: A,alanine; C, cysteine; D aspartic acid; E, glutamic acid; F,phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L,leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R,arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y,tyrosine.

“Polypeptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. A polypeptideis comprised of consecutive amino acids. The term “polypeptide”encompasses naturally occurring or synthetic molecules.

In addition, as used herein, the term “polypeptide” refers to aminoacids joined to each other by peptide bonds or modified peptide bonds,e.g., peptide isosteres, etc. and may contain modified amino acids otherthan the 20 gene-encoded amino acids. The polypeptides can be modifiedby either natural processes, such as post-translational processing, orby chemical modification techniques which are well known in the art.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. The same type of modification can be present in the same orvarying degrees at several sites in a given polypeptide. Also, a givenpolypeptide can have many types of modifications. Modifications include,without limitation, acetylation, acylation, ADP-ribosylation, amidation,covalent cross-linking or cyclization, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphytidylinositol,disulfide bond formation, demethylation, formation of cysteine orpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation, yristolyation,oxidation, pergylation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, and transfer-RNAmediated addition of amino acids to protein such as arginylation. (SeeProteins—Structure and Molecular Properties 2nd Ed., T. E. Creighton,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983)).

As used herein, “peptidomimetic” means a mimetic of a function of aprotein which includes some alteration of the normal peptide chemistry.Peptidomimetics typically are short sequences of amino acids that inbiological properties, mimic one or more function(s) of a particularprotein. Peptide analogs enhance some property of the original peptide,such as increases stability, increased efficacy, enhanced delivery,increased half life, etc. Methods of making peptidomimetics based upon aknown polypeptide sequence is described, for example, in U.S. Pat. Nos.5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involvethe incorporation of a non-amino acid residue with non-amide linkages ata given position. One embodiment of the present invention is apeptidomimetic wherein the compound has a bond, a peptide backbone or anamino acid component replaced with a suitable mimic. Some non-limitingexamples of unnatural amino acids which may be suitable amino acidmimics include β-alanine, L-α-amino butyric acid, L-γ-amino butyricacid, L-α-amino isobutyric acid, L-ε-amino caproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid,N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methioninesulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine,N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine,Boc-hydroxyproline, and Boc-L-thioproline.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combinationthereof.

As used herein, “reverse oriented”, “reversed orientation”, “reverseanalog” or “reverse sequence” refers to a peptide, or a portion of thepeptide, has a reverse amino acid sequence as compared to a non-reverseoriented peptide (i.e., the original sequence is read (or written) fromright to left). For example, if one peptide has the amino acid sequenceABCDE, its reverse analog or a peptide having its reverse sequence is asfollows: EDCBA. In a dual domain peptide for example, Ac-hE-18A-NH₂,either the hE sequence is read from right to left or the 18A sequence isread from right to left. For a reverse analog of,LRKLRKRLLR-DWLKAFYDKVAEKLKEAF can be RLLRKRLKRL-DWLKAFYDKVAEKLKEAF (SEQID NO: 64) or LRKLRKRLLR-FAEKLKEAVKDYFAKLWD (SEQ ID NO: 84).

As used herein a “dual-domain peptide”, a “dual-domain syntheticpeptide”, or a “dual-domain ApoE mimicking peptide” is meant to mean apeptide comprising a lipid-associating peptide/domain and a receptorbinding peptide/domain.

As used herein a “single-domain peptide”, a “single-domain syntheticpeptide”, or a “single-domain ApoE mimicking peptide” is meant to mean apeptide comprising either a lipid-associating peptide/domain or areceptor binding peptide/domain, but not both.

As used herein “domain switched”, “switched domain”, or “switched”peptide is meant to mean that the lipid-associating peptide iscovalently linked to the receptor binding domain of apolipoprotein Esuch that the lipid-associating peptide is at the N-terminus of thesynthetic apolipoprotein E-mimicking peptide. For example, the peptide18A-hE (SEQ ID NO: 38) is exemplary of a domain switched peptide.

As used herein, “scrambled” “scrambled version”, or “scrambled peptide”is meant to mean that the composition of the amino acid sequence is thesame as the unscrambled peptide, however the sequence of the amino acidsis altered thus rendering the peptide unable to form either anα-amphipathic helix or does not possess lipid associating (or HSPGassociating) properties. However, in some cases, as described in thisinvention, the scrambled peptide remains able to form a differenthelical structure, such as a π-helix. For example, if one peptide hasthe amino acid sequence ABCDE, the scrambled version of the peptidecould have the amino acid sequence DEABC. Scrambled peptides are oftendenoted as having an “Sc” prior to the portion of the peptide that isscrambled. For example, Sc-hE-18A denoted that the hE portion of thepeptide is scrambled. FIGS. 21, 23 and 24 show examples of scrambledpeptides.

An “α-amphipathic helix” is discussed above and has 3.6 amino acidresidues per turn of the helix, whereas a “π-helix” has 4.4 amino acidresidues per turn. For example FIGS. 1 and 24 show a difference betweenan “α-amphipathic helix” and a “π-helix”.

As used herein, “sample” is meant to mean an animal; a tissue or organfrom an animal; a cell (either within a subject, taken directly from asubject, or a cell maintained in culture or from a cultured cell line);a cell lysate (or lysate fraction) or cell extract; or a solutioncontaining one or more molecules derived from a cell or cellularmaterial (e.g. a polypeptide or nucleic acid), which is assayed asdescribed herein. A sample may also be any body fluid or excretion (forexample, but not limited to, blood, urine, stool, saliva, tears, bile)that contains cells or cell components.

As used herein, “modulate” is meant to mean to alter, by increasing ordecreasing.

As used herein “lipid binding domain E” and “lipid-associating peptide”are used interchangeably. As used herein, both terms can mean the lipidbinding domain of Apolipoprotein E.

As used herein, “normal subject” is meant to mean an individual who doesnot have a “Lipid Disorder” or an “Inflammatory Disorder”.

As used herein, “Lipid Disorder” is meant to mean when a subject has anexcess of lipids or increased inflammatory lipids in their blood. Lipidsinclude, but are not limited to cholesterol and triglycerides.Inflammatory lipids include, but are not limited to lipids such asox-LDL related lipids (i.e., oxidized PAPC (1-palmitoyl 2-arachidonylphosphatidyl choline). Oxidation of PAPC or PLPC, the lipid componentsof LDL, produce oxidized lipids. Having a lipid disorder can make youmore likely to develop inflammatory diseases such as atherosclerosis andheart disease.

As used herein, “Inflammatory Disorder” is meant to mean when a subjectexperiences a cascade of reactions initiated by oxidized lipids in whichseveral cytokine levels go up to alter the normal physiologicalresponse. Inflammatory disorders include, but are not limited toInflammatory Bowel Disease (IBD), systemic lupus erythematosus,Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease,Sjögren's syndrome, pernicious anemia, Addison disease, Alzheimer'sdisease, scleroderma, Goodpasture's syndrome, ulcerative colitis,Crohn's disease, autoimmune hemolytic anemia, sterility, myastheniagravis, multiple sclerosis, Basedow's disease, thrombopenia purpura,allergy; asthma, atopic disease, arteriosclerosis, myocarditis,cardiomyopathy, glomerular nephritis, hypoplastic anemia, cognition andrejection after organ transplantation. Inflammatory diseases can bebacterial and/or viral in nature.

As used herein, “effective amount” of a compound is meant to mean asufficient amount of the compound to provide the desired effect. Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofdisease (or underlying genetic defect) that is being treated, theparticular compound used, its mode of administration, and the like.Thus, it is not possible to specify an exact “effective amount.”However, an appropriate “effective amount” may be determined by one ofordinary skill in the art using only routine experimentation.

As used herein, “isolated polypeptide” or “purified polypeptide” ismeant to mean a polypeptide (or a fragment thereof) that issubstantially free from the materials with which the polypeptide isnormally associated in nature. The polypeptides of the invention, orfragments thereof, can be obtained, for example, by extraction from anatural source (for example, a mammalian cell), by expression of arecombinant nucleic acid encoding the polypeptide (for example, in acell or in a cell-free translation system), or by chemicallysynthesizing the polypeptide. In addition, polypeptide fragments may beobtained by any of these methods, or by cleaving full length proteinsand/or polypeptides.

As used herein, “isolated nucleic acid” or “purified nucleic acid” ismeant to mean DNA that is free of the genes that, in thenaturally-occurring genome of the organism from which the DNA of theinvention is derived, flank the gene. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector, such asan autonomously replicating plasmid or virus; or incorporated into thegenomic DNA of a prokaryote or eukaryote (e.g., a transgene); or whichexists as a separate molecule (for example, a cDNA or a genomic or cDNAfragment produced by PCR, restriction endonuclease digestion, orchemical or in vitro synthesis). It also includes a recombinant DNAwhich is part of a hybrid gene encoding additional polypeptide sequence.The term “isolated nucleic acid” also refers to RNA, e.g., an mRNAmolecule that is encoded by an isolated DNA molecule, or that ischemically synthesized, or that is separated or substantially free fromat least some cellular components, for example, other types of RNAmolecules or polypeptide molecules.

As used herein, “transgene” is meant to man a nucleic acid sequence thatis inserted by artifice into a cell and becomes a part of the genome ofthat cell and its progeny. Such a transgene may be (but is notnecessarily) partly or entirely heterologous (for example, derived froma different species) to the cell.

As used herein, “transgenic animal” is meant to mean an animalcomprising a transgene as described above. Transgenic animals are madeby techniques that are well known in the art.

As used herein, “knockout mutation” is meant to mean an alteration inthe nucleic acid sequence that reduces the biological activity of thepolypeptide normally encoded therefrom by at least 80% relative to theunmutated gene. The mutation may, without limitation, be an insertion,deletion, frameshift, or missense mutation. A “knockout animal,” forexample, a knockout mouse, is an animal containing a knockout mutation.The knockout animal may be heterozygous or homozygous for the knockoutmutation. Such knockout animals are generated by techniques that arewell known in the art.

As used herein, “treat” is meant to mean administer a compound ormolecule of the invention to a subject, such as a human or other mammal(for example, an animal model), that has a Lipid Disorder, or that hascoronary artery disease, rheumatoid arthritis, and/or systemic lupus, inorder to prevent or delay a worsening of the effects of the disease orcondition, or to partially or fully reverse the effects of the disease.

As used herein, “prevent” is meant to mean minimize the chance that asubject who has an increased susceptibility for developing a LipidDisorder will develop a Lipid Disorder.

As used herein, “specifically binds” is meant that an antibodyrecognizes and physically interacts with its cognate antigen (forexample, the disclosed synthetic apolipoprotein E-mimicking peptides)and does not significantly recognize and interact with other antigens;such an antibody may be a polyclonal antibody or a monoclonal antibody,which are generated by techniques that are well known in the art.

As used herein, “probe,” “primer,” or oligonucleotide is meant to mean asingle-stranded DNA or RNA molecule of defined sequence that canbase-pair to a second DNA or RNA molecule that contains a complementarysequence (the “target”). The stability of the resulting hybrid dependsupon the extent of the base-pairing that occurs. The extent ofbase-pairing is affected by parameters such as the degree ofcomplementarity between the probe and target molecules and the degree ofstringency of the hybridization conditions. The degree of hybridizationstringency is affected by parameters such as temperature, saltconcentration, and the concentration of organic molecules such asformamide, and is determined by methods known to one skilled in the art.Probes or primers specific for nucleic acids capable of encoding thedisclosed synthetic apolipoprotein E-mimicking peptide (for example,genes and/or mRNAs) have at least 80%-90% sequence complementarity,preferably at least 91%-95% sequence complementarity, more preferably atleast 96%-99% sequence complementarity, and most preferably 100%sequence complementarity to the region of the nucleic acid capable ofencoding the disclosed synthetic apolipoprotein E-mimicking peptide towhich they hybridize. Probes, primers, and oligonucleotides may bedetectably-labeled, either radioactively, or non-radioactively, bymethods well-known to those skilled in the art. Probes, primers, andoligonucleotides are used for methods involving nucleic acidhybridization, such as: nucleic acid sequencing, reverse transcriptionand/or nucleic acid amplification by the polymerase chain reaction,single stranded conformational polymorphism (SSCP) analysis, restrictionfragment polymorphism (RFLP) analysis, Southern hybridization, Northernhybridization, in situ hybridization, electrophoretic mobility shiftassay (EMSA).

As used herein, “specifically hybridizes” is meant to mean that a probe,primer, or oligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a nucleic acid capable of encoding the disclosed syntheticapolipoprotein E-mimicking peptide) under high stringency conditions,and does not substantially base pair with other nucleic acids.

As used herein, “high stringency conditions” is meant to mean conditionsthat allow hybridization comparable with that resulting from the use ofa DNA probe of at least 40 nucleotides in length, in a buffer containing0.5 M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well-known by thoseskilled in the art of molecular biology. (See, for example, F. Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1998).

As used herein, “lipoprotein” or “lipoproteins” is meant to mean abiochemical assembly that contains both proteins and lipids. The lipidsor their derivatives may be covalently or non-covalently bound to theproteins. Many enzymes, transporters, structural proteins, antigens,adhesins, and toxins are lipoproteins. Examples include the high densityand low density lipoproteins of the blood, the transmembrane proteins ofthe mitochondrion and the chloroplast, and bacterial lipoproteins

As used herein, “high-density lipoprotein” (HDL) is meant to mean aclass of lipoproteins, varying somewhat in their size (8-11 nm indiameter), that can transport cholesterol.

As used herein, “very Low Density Lipoproteins” (VLDL) is meant to meana lipoprotein subclass. It is assembled in the liver from cholesteroland apolipoproteins. It is converted in the bloodstream to low densitylipoprotein (LDL). VLDL particles have a diameter of 30-80 nm. VLDLtransports endogenous products where chylomicrons transport exogenous(dietary) products.

As used herein, “low-density lipoprotein” or “LDL” is mean to mean alipoprotein that varies in size (approx. 22 nm) and can contain achanging number of fatty acids they actually have a mass and sizedistribution. Each native LDL particle contains a singleapolipoproteinapolipoprotein B-100 molecule (Apo B-100, a protein with4536 amino acidamino acid residues) that circles the fatty acids keepingthem soluble in the aquous environment. LDL is commonly referred to asbad cholesterol

Cholesterol cannot dissolve in the blood. It has to be transported toand from the cells by carriers called lipoproteins. LDLs and HDLs alongwith triglyceride-rich lipoproteins (VLDL) and Lp(a) cholesterol, makeup your total cholesterol count, which can be determined through a bloodtest.

As used herein, “LDL cholesterol” is meant to mean cholesterol that isassociated with LDLs. When too much LDL cholesterol circulates in theblood, it can slowly build up in the inner walls of the arteries thatfeed the heart and brain. Together with other substances, it can formplaque, a thick, hard deposit that can narrow the arteries and make themless flexible. This condition is known as atherosclerosis. If a clotforms and blocks a narrowed artery, then heart attack or stroke canresult.

As used herein, “VLDL cholesterol” is meant to mean cholesterol that isassociated with VLDLs.

As used herein, “HDL cholesterol” is meant to mean cholesterol that isassociated with HDLs. About one-fourth to one-third of blood cholesterolis carried by high-density lipoprotein (HDL). HDL cholesterol is knownas “good” cholesterol, because high levels of HDL seem to protectagainst heart attack. Low levels of HDL (less than 40 mg/dL in men andless than 50 mg/dL in women) also increase the risk of heart disease.Medical experts think that HDL tends to carry cholesterol away from thearteries and back to the liver, where it is passed from the body. Someexperts believe that that HDL removes excess cholesterol from arterialplaque, thus slowing its buildup.

As used herein, “Lp(a)” is meant to mean a genetic variation of LDL(bad) cholesterol. A high level of Lp(a) is a significant risk factorfor the premature development of fatty deposits in arteries. Lp(a) isnot fully understood, but it may interact with substances found inartery walls and contribute to the buildup of fatty deposits.

B. COMPOUNDS AND COMPOSITIONS OF THE INVENTION

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein.

Peptides

Human apolipoprotein E (apo E) consists of two distinct domains, thelipid-associating domain (residues 192-299) and the globular domain(1-191) which contains the LDL receptor binding site (residues 129-169).To test the hypothesis that a minimal arginine-rich apoE receptorbinding domain (141-150) was sufficient to enhance low densitylipoprotein (LDL) and very low density lipoprotein (VLDL) uptake andclearance when covalently linked to a class A amphipathic helix,Anantharamaiah et al. synthesized a peptide in which the receptorbinding domain of human apo E, LRKLRKRLLR (hApo E[141-150] also referredto as “hE”, SEQ ID NO: 1), was linked to 18A, a well characterized highaffinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF, also referred toas “18A”, SEQ ID NO: 4) to produce a peptide denoted ashApoE[141-150]-18A (also referred to as “hE-18A”, SEQ ID NO: 11) (seeU.S. Pat. No. 6,506,880, which is hereby incorporated by reference inits entirety for its teaching of specific apolipoprotein E-mimickingpeptides and their uses). Also synthesiszed was an end protected analogof hE-18A, denoted Ac-hE18A-NH₂(SEQ ID NO: 12). The importance of thelysine residues and the role of the hydrophobic residues in the receptorbinding domain were also studied using two analogs, LRRLRRRLLR-18A (alsoreferred to as “hE(R)-18A”, SEQ ID NO: 13) and LRKMRKRLMR-18A (alsoreferred to as “mE18A”, SEQ ID NO: 14), whereby the receptor bindingdomain of human apo E was modified to substitute arginine (R) residuesfor lysine (K) residues at positions 143 and 146 (SEQ ID NO: 3) andwhereby the receptor binding domain of mouse apo E (SEQ ID NO: 2), werelinked to 18A, respectively. The effect of the dual character peptideson the uptake and degradation of human LDL/VLDL by cells was thendetermined.

It was determined that in MEF 1 cells with induced LDL receptors, LDLinternalization was enhanced three, five and seven times byAc-mE-18A-NH₂, Ac-hE-18A-NH₂, and Ac-hE(R)-18A-NH₂ respectively. Allthree peptides increased degradation of LDL by 100 percent. BothAc-hE-18A-NH₂ and the control peptide Ac-18A-NH₂ interacted with VLDL tocause a displacement of apo E from VLDL. However, onlyAc-hE-18A-NH₂-associated VLDL enhanced the uptake of VLDL six fold anddegradation three fold compared to VLDL alone in spite of the absence ofapo E. The LDL binding to fibroblasts in the presence of these peptideswas not saturable, however, over the LDL concentration range studied.

Furthermore, Anantharamaiah et al. showed a similar enhancement of LDLinternalization independent of the presence of the LDL receptor relatedprotein (LRP) or LDL receptor or both. Pretreatment of cells withheparinase and heparitinase however abolished greater than 80% ofenhanced peptide-mediated LDL uptake and degradation by cells. The dataindicated that the dual-domain peptides enhanced LDL uptake anddegradation by binding to the LDL through the amphipathic lipid bindingdomain (18A). However, the minimal 141-150 Arg-rich domain did notdecrease LDL levels but did so only in combination with 18A lipidassociating domain, did not confer LDL-receptor binding but directed theLDL-peptide complex to the HSPG pathway for uptake and degradation byfibroblasts.

Non-Limiting Examples of Polypeptides and Peptides of the Invention

The present invention is directed to a synthetic apolipoprotein-Emimicking peptide or polypeptide. Non-limiting examples of the syntheticapolipoprotein-E mimicking peptides or polypeptides of the invention aregiven below. Disclosed herein are synthetic apolipoprotein E-mimickingpeptides, consisting of: a receptor binding domain of apolipoprotein Ecomprising the amino acid sequence of SEQ ID NO: 15; and alipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide. As such, thereceptor binding domain replaced the two leucine (L) residues atpositions 148 and 149 of LRKLRKRLLR (hApo E[141-150], SEQ ID NO: 1) withtwo phenylalanine (F) residues. The lipid associating peptide for thesesynthetic apolipoprotein E-mimicking peptides can be the model class Aamphipathic helical peptide 18A. For example the lipid-associatingpeptide can comprise the amino acid sequence of SEQ ID NO: 16 or SEQ IDNO: 17.

Also disclosed herein are synthetic apolipoprotein E-mimicking peptides,comprising: a lipid binding domain of apolipoprotein E comprising theamino acid sequence of SEQ ID NO: 17; and a receptor binding domainpeptide, wherein said lipid binding domain is covalently linked to saidreceptor binding domain peptide. As such, the lipid binding domainreplaced the two leucine (L) residues of DWLKAFYDKVAEKLKEAF (18A, SEQ IDNO: 16) with two phenylalanine (F) residues resulting in the sequenceDWFKAFYDKVAEKFKEAF (SEQ ID NO: 17, also referred to as modified 18A orm18A). The receptor binding domain peptide for the syntheticapolipoprotein E-mimicking peptides can be a human receptor bindingdomain peptide of ApoE. For example, receptor binding domain peptide ofthe disclosed synthetic apolipoprotein E-mimicking peptides can comprisethe amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 15.The receptor binding domain peptide of such synthetic apolipoproteinE-mimicking peptides can also be from a species selected from the groupconsisting of mouse, rabbit, monkey, rat, bovine, pig and dog.

The receptor binding domain peptide for the synthetic apolipoproteinE-mimicking peptides can also be the LDL receptor (LDLR) binding domainof apolipoprotein B (ApoB). The LDL receptor (LDLR) binding domain ofApoB can have the sequence RLTRKRGLK (SEQ ID NO. 104). ApoB-100 is a550,000 Da glycoprotein with nine amino acids (3359-3367) serving as thebinding domain for the LDL receptor (Segrest et al., J. Lipid. Res. 42,pp. 1346-1367 (2001)). Upon binding to LDLR in clathrin coated pits, LDLis internalized via endocytosis and moves into the endosome where a dropin pH causes the receptor to dissociate from the LDL. The receptor isrecycled back to the surface of the cell while the LDL is moved into thelysosome where the particle is degraded (Goldstein et al., Ann. Rev.Cell Biol. 1, pp. 1-39 (1985)). The LDL receptor (LDLR) binding domainof ApoB when used with the disclosed peptides can also be altered and/ormodified as described throughout this application for ApoE. For example,LDL receptor (LDLR) binding domain of ApoB can be used with thedisclosed lipid-associating peptides, wherein the LDL receptor (LDLR)binding domain of ApoB is covalently linked to said lipid-associatingpeptide. In addition, the LDL receptor (LDLR) binding domain of ApoB canbe scrambled, reverse-oriented, can be part of a domain switched peptideas described below.

Examples of receptor binding domain peptides that can be used in thedisclosed synthetic apolipoprotein E-mimicking peptides are provided inTable 1.

TABLE 1 Disclosed Synthetic Apolipoprotein E-Mimicking Peptides StartingSpecies Residue NO: Sequence SEQ ID NO: Human 141 LRKLRKRLLRSEQ ID NO: 1 Rabbit 134 LRKLRKRLLR SEQ ID NO: 5 Monkey 141 LRKLRKRLLRSEQ ID NO: 6 Mouse 133 LRKMRKRL M R SEQ ID NO: 2 Rat 133 LRKMRKRL M RSEQ ID NO: 7 Bovine 140 LRKL

KRLLR SEQ ID NO: 8 Pig 140 LR NVRKRL V R SEQ ID NO: 9 Dog 133 MRKLRKRVLRSEQ ID NO: 10 R Modified 141 LR RLRR RLLR SEQ ID NO: 3 F Modified 141LRKLRKR

R SEQ ID NO: 15 ApoB

SEQ ID NO: 104

The italicized residues in Table 1 indicate changes from the humansequence; however, the property of the amino acid is conserved. Thebold-italicized residues in Table 1 indicate the difference from thehuman sequence at that position.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of a combination of the disclosed receptor binding domains ofapolipoprotein E and the disclosed lipid-associating peptides, whereinsaid receptor binding domain is covalently linked to saidlipid-associating peptide. Additional lipid-associating peptides thatcan be used in the disclosed compositions are described in U.S. patentapplication Ser. No. 11/407,390 (Fogelman et al.), which is herebyincorporated by reference in its entirety for its teaching oflipid-associating peptides. For example, the lipid-associating peptidesof Tables 2-6 of U.S. patent application Ser. No. 11/407,390 can be usedin the disclosed compositions.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of a combination of the disclosed receptor binding domains ofapolipoprotein B and the disclosed lipid-associating peptides, whereinsaid receptor binding domain is covalently linked to saidlipid-associating peptide. Non-limiting examples of the disclosedsynthetic apolipoprotein E-mimicking peptides are provided in Table 2.The disclosed synthetic apolipoprotein E-mimicking peptides can also beN-terminally protected using acetyl and amino groups.

TABLE 2 Non-limiting Examples of the Disclosed SyntheticApolipoprotein E-Mimicking Peptides Receptor Binding Lipid-AssociatingDomains of ApoE Peptides SEQ ID NO: LRKLRKRLLR DWLKAFYDKVAEKLKEAFSEQ ID NO: 18 LRKLRKRLLR DWLKAFYDKVAEKLKEAF SEQ ID NO: 19 LRKLRKRLLRDWLKAFYDKVAEKLKEAF SEQ ID NO: 20 LRKMRKRL M R DWLKAFYDKVAEKLKEAFSEQ ID NO: 21 LRKMRKRL M R DWLKAFYDKVAEKLKEAF SEQ ID NO: 22 LRKL

KRLLR DWLKAFYDKVAEKLKEAF SEQ ID NO: 23 LR NVRKRL V R DWLKAFYDKVAEKLKEAFSEQ ID NO: 24 MRKLRKRVLR DWLKAFYDKVAEKLKEAF SEQ ID NO: 25 LR RLRR RLLRDWLKAFYDKVAEKLKEAF SEQ ID NO: 26 LRKLRKR

R DWLKAFYDKVAEKLKEAF SEQ ID NO: 27 LRKLRKRLLR DWFKAFYDKVAEKFKEAFSEQ ID NO: 28 LRKLRKRLLR DWFKAFYDKVAEKFKEAF SEQ ID NO: 29 LRKLRKRLLRDWFKAFYDKVAEKFKEAF SEQ ID NO: 30 LRKMRKRL M R DWFKAFYDKVAEKFKEAFSEQ ID NO: 31 LRKMRKRL M R DWFKAFYDKVAEKFKEAF SEQ ID NO: 32 LRKL

KRLLR DWFKAFYDKVAEKFKEAF SEQ ID NO: 33 LR NVRKRL V R DWFKAFYDKVAEKFKEAFSEQ ID NO: 34 MRKLRKRVLR DWFKAFYDKVAEKFKEAF SEQ ID NO: 35 LR RLRR RLLRDWFKAFYDKVAEKFKEAF SEQ ID NO: 36 LRKLRKR

R DWFKAFYDKVAEKFKEAF SEQ ID NO: 37

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of a combination of the disclosed receptor binding domains ofapolipoprotein E and the disclosed lipid-associating peptides, whereinsaid receptor binding domain is covalently linked to saidlipid-associating peptide in a domain switched orientation. Alsodisclosed are synthetic apolipoprotein E-mimicking peptides, consistingof a combination of the disclosed receptor binding domains ofapolipoprotein B and the disclosed lipid-associating peptides, whereinsaid receptor binding domain is covalently linked to saidlipid-associating peptide in a domain switched orientation. Thesepeptides can be referred to as “domain switched” “switched domain”, or“switched” peptides. For example, disclosed are synthetic apolipoproteinE-mimicking peptides, consisting of a combination of the disclosedreceptor binding domains of apolipoprotein E and the disclosedlipid-associating peptides, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide in a domain switchedorientation to those described above and in Table 2. Specifically, thelipid-associating peptide is covalently linked to the receptor bindingdomain of apolipoprotein E such that the lipid-associating peptide is atthe N-terminus of the synthetic apolipoprotein E-mimicking peptide.Non-limiting examples of the disclosed synthetic apolipoproteinE-mimicking peptides are provided in Table 3.

TABLE 3 Non-limiting Examples of Disclosed SyntheticApolipoprotein E-Mimicking Peptides Lipid-Associating Receptor BindingPeptides Domains of ApoE SEQ ID NO: DWLKAFYDKVAEKLKEAF LRKLRKRLLRSEQ ID NO: 38 DWLKAFYDKVAEKLKEAF LRKLRKRLLR SEQ ID NO: 39DWLKAFYDKVAEKLKEAF LRKLRKRLLR SEQ ID NO: 40 DWLKAFYDKVAEKLKEAF LRKMRKRLM R SEQ ID NO: 41 DWLKAFYDKVAEKLKEAF LRKMRKRL M R SEQ ID NO: 42DWLKAFYDKVAEKLKEAF LRKL

KRLLR SEQ ID NO: 43 DWLKAFYDKVAEKLKEAF LR NVRKRLVR SEQ ID NO: 44DWLKAFYDKVAEKLKEAF MRKLRKRVLR SEQ ID NO: 45 DWLKAFYDKVAEKLKEAF LR RLRRRLLR SEQ ID NO: 46 DWLKAFYDKVAEKLKEAF LRKLRKR

R SEQ ID NO: 47 DWFKAFYDKVAEKFKEAF LRKLRKRLLR SEQ ID NO: 48DWFKAFYDKVAEKFKEAF LRKLRKRLLR SEQ ID NO: 49 DWFKAFYDKVAEKFKEAFLRKLRKRLLR SEQ ID NO: 50 DWFKAFYDKVAEKFKEAF LRKMRKRL M R SEQ ID NO: 51DWFKAFYDKVAEKFKEAF LRKMRKRL M R SEQ ID NO: 52 DWFKAFYDKVAEKFKEAF LRKL

KRLLR SEQ ID NO: 53 DWFKAFYDKVAEKFKEAF LR NVRKRL V R SEQ ID NO: 54DWFKAFYDKVAEKFKEAF MRKLRKRVLR SEQ ID NO: 55 DWFKAFYDKVAEKFKEAF LR RLRRRLLR SEQ ID NO: 56 DWFKAFYDKVAEKFKEAF LRKLRKR

R SEQ ID NO: 57

The disclosed domain switched synthetic apolipoprotein E-mimickingpeptides can also be N-terminally protected using acetyl and aminogroups.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of a combination of the disclosed receptor binding domains ofapolipoprotein E and the disclosed lipid-associating peptides, whereinsaid receptor binding domain is covalently linked to saidlipid-associating peptide in a reversed orientation. For example,disclosed are synthetic apolipoprotein E-mimicking peptides, consistingof a combination of the disclosed receptor binding domains ofapolipoprotein E and the disclosed lipid-associating peptides, whereineither the sequence of the receptor binding domain or the sequence ofthe lipid-associating peptide or both sequences are in the reversedoritentation. Also disclosed are synthetic apolipoprotein E-mimickingpeptides, consisting of a combination of the disclosed receptor bindingdomains of apolipoprotein B and the disclosed lipid-associatingpeptides, wherein said receptor binding domain is covalently linked tosaid lipid-associating peptide in a reversed orientation. Non-limitingexamples of the disclosed synthetic apolipoprotein E-mimicking peptidesare provided in Table 4.

TABLE 4 Non-limiting Examples of SyntheticApolipoprotein E-Mimicking Peptides Receptor Binding Lipid-AssociatingDomains of ApoE Peptides SEQ ID NO: RLLRKRLKRL DWLKAFYDKVAEKLKEAFSEQ ID NO: 64 RLLRKRLKRL DWLKAFYDKVAEKLKEAF SEQ ID NO: 65 RLLRKRLKRLDWLKAFYDKVAEKLKEAF SEQ ID NO: 66 RMLRKRMKRL DWLKAFYDKVAEKLKEAFSEQ ID NO: 67 RMLRKRMKRL DWLKAFYDKVAEKLKEAF SEQ ID NO: 68 RLLRKPLKRLDWLKAFYDKVAEKLKEAF SEQ ID NO: 69 RVLRKRVNRL DWLKAFYDKVAEKLKEAFSEQ ID NO: 70 RLVRKRLKRM DWLKAFYDKVAEKLKEAF SEQ ID NO: 71 RLLRRRLRRLDWLKAFYDKVAEKLKEAF SEQ ID NO: 72 RFFRKRLKRL DWLKAFYDKVAEKLKEAFSEQ ID NO: 73 RLLRKRLKRL DWFKAFYDKVAEKFKEAF SEQ ID NO: 74 RLLRKRLKRLDWFKAFYDKVAEKFKEAF SEQ ID NO: 75 RLLRKRLKRL DWFKAFYDKVAEKFKEAFSEQ ID NO: 76 RMLRKRMKRL DWFKAFYDKVAEKFKEAF SEQ ID NO: 77 RMLRKRMKRLDWFKAFYDKVAEKFKEAF SEQ ID NO: 78 RLLRKPLKRL DWFKAFYDKVAEKFKEAFSEQ ID NO: 79 RVLRKRVNRL DWFKAFYDKVAEKFKEAF SEQ ID NO: 80 RLVRKRLKRMDWFKAFYDKVAEKFKEAF SEQ ID NO: 81 RLLRRRLRRL DWFKAFYDKVAEKFKEAFSEQ ID NO: 82 RFFRKRLKRL DWFKAFYDKVAEKFKEAF SEQ ID NO: 83 LRKLRKRLLRFAEKLKEAVKDYFAKLWD SEQ ID NO: 84 LRKLRKRLLR FAEKLKEAVKDYFAKLWDSEQ ID NO: 85 LRKLRKRLLR FAEKLKEAVKDYFAKLWD SEQ ID NO: 86 LRKMRKRLMRFAEKLKEAVKDYFAKLWD SEQ ID NO: 87 LRKMRKRLMR FAEKLKEAVKDYFAKLWDSEQ ID NO: 88 LRKLPKRLLR FAEKLKEAVKDYFAKLWD SEQ ID NO: 89 LRNVRKRLVRFAEKLKEAVKDYFAKLWD SEQ ID NO: 90 MRKLRKRVLR FAEKLKEAVKDYFAKLWDSEQ ID NO: 91 LRRLRRRLLR FAEKLKEAVKDYFAKLWD SEQ ID NO: 92 LRKLRKRFFRFAEKLKEAVKDYFAKLWD SEQ ID NO: 93 LRKLRKRLLR FAEKFKEAVKDYFAKFWDSEQ ID NO: 94 LRKLRKRLLR FAEKFKEAVKDYFAKFWD SEQ ID NO: 95 LRKLRKRLLRFAEKFKEAVKDYFAKFWD SEQ ID NO: 96 LRKMRKRLMR FAEKFKEAVKDYFAKFWDSEQ ID NO: 97 LRKMRKRLMR FAEKFKEAVKDYFAKFWD SEQ ID NO: 98 LRKLPKRLLRFAEKFKEAVKDYFAKFWD SEQ ID NO: 99 LRNVRKRLVR FAEKFKEAVKDYFAKFWDSEQ ID NO: 100 MRKLRKRVLR FAEKFKEAVKDYFAKFWD SEQ ID NO: 101 LRRLRRRLLRFAEKFKEAVKDYFAKFWD SEQ ID NO: 102 LRKLRKRFFR FAEKFKEAVKDYFAKFWDSEQ ID NO: 103

The disclosed reverse-oriented synthetic apolipoprotein E-mimickingpeptides can also be N-terminally and C-terminally protected usingacetyl and amide groups.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide, wherein thereceptor binding domain of apolipoprotein E is scrambled. For example,disclosed is a synthetic apolipoprotein E-mimicking peptide, consistingof: a receptor binding domain of apolipoprotein E comprising the aminoacid sequence of SEQ ID NO: 58; and a lipid-associating peptide, whereinsaid receptor binding domain is covalently linked to saidlipid-associating peptide. Also disclosed are synthetic apolipoproteinE-mimicking peptides, consisting of: a receptor binding domain ofapolipoprotein B and a lipid-associating peptide, wherein said receptorbinding domain is covalently linked to said lipid-associating peptide,wherein the receptor binding domain of apolipoprotein B is scrambled.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide, wherein thelipid-associating peptide is scrambled. For example, disclosed herein isa synthetic apolipoprotein E-mimicking peptides, comprising: a lipidbinding domain of apolipoprotein E comprising the amino acid sequence ofSEQ ID NO: 59 and a receptor binding domain peptide, wherein said lipidbinding domain is covalently linked to said receptor binding domainpeptide.

Also disclosed are synthetic apolipoprotein E-mimicking peptides,consisting of: a receptor binding domain of apolipoprotein E and alipid-associating peptide of apolipoprotein E, wherein receptor bindingdomain is covalently linked to said lipid-associating peptide, whereinboth the receptor binding domain and the lipid-associating peptide arescrambled. Non-limiting examples of the disclosed scrambled syntheticapolipoprotein E-mimicking peptides are provided in Table 5.

TABLE 5  Scrambled Synthetic Apoliprotein E-Mimicking PeptidesReceptor Binding Lipid-Associating Name Domains of ApoE PeptidesSEQ ID NO: hE-Sc 18A LRKLRKRLLR KAFEEVLAKKFYDKALWD SEQ ID NO: 60(hE with Sc18A also referred to as Sc2F) SchE-18A LRLLRKLKRRDWLKAFYDKVAEKLKEAF SEQ ID NO: 61

The disclosed scrambled synthetic apolipoprotein E-mimicking peptidescan also be N-terminally and C-terminally protected using acetyl andamide groups. The disclosed scrambled synthetic apolipoproteinE-mimicking peptides can also be reverse-oriented as described above.

Also disclosed are single-domain synthetic apolipoprotein E-mimickingpeptides. The single-domain synthetic apolipoprotein E-mimickingpeptides can consist of a receptor binding domain of apolipoprotein E ora lipid-associating peptide. The receptor binding domain or thelipid-associating peptide can be modified or altered as described above.For example, the receptor binding domain or the lipid-associatingpeptide can be mutated, scrambeled, and/or reverse-oriented. Any othermodifications or alterations disclosed herein for the dual-domainpolypeptides can also be used for the single-domain peptides. Numerousother variants or derivatives of the peptides disclosed herein are alsocontemplated. For example, scrambled peptides can also bereverse-oriented, or can be in a switched orientation. Additionally,reverse-oriented peptides can be in a switched orientation. All othercombinations of the disclosed peptides are also contemplated.Non-limiting examples of the peptides have been described herein (seeTables 1-5, for example). As used herein, the term “analog” is usedinterchangeably with “variant” and “derivative.” Variants andderivatives are well understood to those of skill in the art and caninvolve amino acid sequence modifications. Such, amino acid sequencemodifications typically fall into one or more of three classes:substantial; insertional; or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily are smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.These variants ordinarily are prepared by site-specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis and PCR mutagenesis. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once. Substitutions, deletions,insertions or any combination thereof may be combined to arrive at afinal derivative or analog. Substutitional variants are those in whichat least one residue has been removed and a different residue insertedin its place. Such substitutions generally are made in accordance withTables 6 and 7 and are referred to as conservative substitutions.

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table6, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties are those in which: (a) the hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; Tryptophan, Tyrosinyl(b) a cysteine or proline is substituted for (or by) any other residue;(c) a residue having an electropositive side chain, e.g., lysyl,arginyl, or hystidyl, is substituted for (or by) an electronegativeresidue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky sidechain, e.g., phenylalanine, is substituted for (or by) one not having aside chain, e.g., glycine, in this case, or (e) by increasing the numberof sites for sulfation and/or glycosylation.

TABLE 6 Amino Acid Substitutions Non-limiting Exemplary Original ResidueConservative Substitutions Ala Ser Arg Gly; Gln; Lys Asn Gln; His AspGlu Cys Ser Gln Asn; Lys Glu Asp Gly Ala His Asn; Gln Ile Leu; Val LeuIle; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser TrpTyr Tyr Trp; Phe Val Ile; Leu

TABLE 7 Amino Acid Abbreviations Amino Acid Abbreviations Alanine Ala(A) Allosoleucine AIle Arginine Arg (R) Asparagine Asn (N) Aspartic AcidAsp (D) Cysteine Cys (C) Glutamic Acid Glu (E) Glutamine Gln (Q) GlycineGly (G) Histidine His (H) Isolelucine Ile (I) Leucine Leu (L) Lysine Lys(K) Phenylalanine Phe (F) Praline Pro (P) Pyroglutamic Acid PGlu (U)Serine Ser (S) Threonine Thr (T) Tyrosine Tyr (Y) Tryptophan Trp (W)Valine Val (V)

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is to define them in terms ofhomology/identity to specific known sequences. Specifically disclosedare variants of synthetic apolipoprotein E-mimicking peptides and otherproteins or peptides herein disclosed which have at least, 70% or atleast 75% or at least 80% or at least 85% or at least 90% or at least95% homology to the synthetic apolipoprotein E-mimicking peptidesspecifically recited herein. Those of skill in the art readilyunderstand how to determine the homology of two proteins.

As this specification discusses various polypeptides and polypeptidesequences it is understood that the nucleic acids that can encode thosepolypeptide sequences are also disclosed. This would include alldegenerate sequences related to a specific polypeptide sequence, i.e.all nucleic acids having a sequence that encodes one particularpolypeptide sequence as well as all nucleic acids, including degeneratenucleic acids, encoding the disclosed variants and derivatives of theprotein sequences. Thus, while each particular nucleic acid sequence maynot be written out herein, it is understood that each and every sequenceis in fact disclosed and described herein through the disclosedpolypeptide sequences.

Blocking/Protecting Groups and D Residues

While the various compositions described herein may be shown with noprotecting groups, in certain embodiments (e.g., particularly for oraladministration), they can bear one, two, three, four, or more protectinggroups. The protecting groups can be coupled to the C- and/or N-terminusof the peptide(s) and/or to one or more internal residues comprising thepeptide(s) (e.g., one or more R-groups on the constituent amino acidscan be blocked). Thus, for example, in certain embodiments, any of thepeptides described herein can bear, e.g., an acetyl group protecting theamino terminus and/or an amide group protecting the carboxyl terminus.One example of such a “dual protected peptide” isAc-LRKLRKRLLRDWLKAFYDKVAEKLKEAF—NH₂ (SEQ ID NO:12 with blocking groups),either or both of these protecting groups can be eliminated and/orsubstituted with another protecting group as described herein. Withoutbeing bound by a particular theory, it was a discovery of this inventionthat blockage, particularly of the amino and/or carboxyl termini of thesubject peptides of this invention can improve oral delivery and canalso increase serum half-life.

A wide number of protecting groups are suitable for this purpose. Suchgroups include, but are not limited to acetyl, amide, and alkyl groupswith acetyl and alkyl groups being particularly preferred for N-terminalprotection and amide groups being preferred for carboxyl terminalprotection. For example, the protecting groups can include, but are notlimited to alkyl chains as in fatty acids, propeonyl, formyl, andothers. Carboxyl protecting groups include amides, esters, andether-forming protecting groups can also be used. For example, an acetylgroup can be used to protect the amino terminus and an amide group canbe used to protect the carboxyl terminus. These blocking groups enhancethe helix-forming tendencies of the peptides. Additional blocking groupsinclude alkyl groups of various lengths, e.g., groups having theformula: CH₃(CH₂)_(n)CO where n ranges from about 1 to about 20,preferably from about 1 to about 16 or 18, more preferably from about 3to about 13, and most preferably from about 3 to about 10.

Additionally, the protecting groups include, but are not limited toalkyl chains as in fatty acids, propeonyl, formyl, and others. Forexample, carboxyl protecting groups can include amides, esters, andether-forming protecting groups. These blocking groups can enhance thehelix-forming tendencies of the peptides. Blocking groups can includealkyl groups of various lengths, e.g., groups having the formula:CH₃(CH₂)_(n)CO where n ranges from about 3 to about 20, preferably fromabout 3 to about 16, more preferably from about 3 to about 13, and mostpreferably from about 3 to about 10.

Other protecting groups include, but are not limited to Fmoc,t-butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylicgroup, 9-florenecarboxylic group, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh)Tosyl (Tos),2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl),4-methoxybenzyl (MeOBzl), Benzyloxy (Bz10), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl(2-Cl—Z),2-bromobenzyloxy-carbonyl (2-Br-Z), Benzyloxymethyl (Bom),cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl(tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

Protecting/blocking groups are well known to those of skill as aremethods of coupling such groups to the appropriate residue(s) comprisingthe peptides of this invention (see, e.g., Greene et al., (1991)Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc.Somerset, N.J.). For example, acetylation can be accomplished during thesynthesis when the peptide is on the resin using acetic anhydride. Amideprotection can be achieved by the selection of a proper resin for thesynthesis.

The compositions disclosed herein can also comprise one or more D-form(dextro rather than levo) amino acids as described herein. For example,at least two enantiomeric amino acids, at least 4 enantiomeric aminoacids or at least 8 or 10 enantiomeric amino acids can be in the “D”form amino acids. Additionally, every other, or even every amino acid(e.g., every enantiomeric amino acid) of the peptides described hereinis a D-form amino acid.

Additionally, at least 50% of the enantiomeric amino acids can be “D”form, at least 80% of the enantiomeric amino acids are “D” form, atleast 90%, or even all of the enantiomeric amino acids can be in the “D”form amino acids.

Polypeptide Production

Polypeptides of the invention are produced by any method known in theart. One method of producing the disclosed polypeptides is to link twoor more amino acid residues, peptides or polypeptides together byprotein chemistry techniques. For example, peptides or polypeptides arechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry (Applied Biosystems, Inc., FosterCity, Calif.). A peptide or polypeptide can be synthesized and notcleaved from its synthesis resin, whereas the other fragment of apeptide or protein can be synthesized and subsequently cleaved from theresin, thereby exposing a terminal group, which is functionally blockedon the other fragment. By peptide condensation reactions, these twofragments can be covalently joined via a peptide bond at their carboxyland amino termini, respectively, (Grant G A (1992) Synthetic Peptides: AUser Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B.,Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY).Alternatively, the peptide or polypeptide is independently synthesizedin vivo. Once isolated, these independent peptides or polypeptides maybe linked to form a peptide or fragment thereof via similar peptidecondensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two-step chemical reaction (Dawson et al.Science, 266:776-779 (1994)). The first step is the chemoselectivereaction of an unprotected synthetic peptide-thioester with anotherunprotected peptide segment containing an amino-terminal Cys residue togive a thioester-linked intermediate as the initial covalent product.Without a change in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site (Baggiolim M et al. (1992) FEBS Lett. 307:97 -101;Clark-Lewis I et al., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I etal., Biochem., 30:3128 (1991); Rajarathnam K et al., Biochem. 33:6623-30(1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

Antibodies

Also disclosed herein are isolated antibodies, antibody fragments andantigen-binding fragments thereof, that specifically bind to one or moreof the synthetic apolipoprotein E-mimicking peptides disclosed herein.Optionally, the isolated antibodies, antibody fragments, orantigen-binding fragment thereof can be neutralizing antibodies. Theantibodies, antibody fragments and antigen-binding fragments thereofdisclosed herein can be identified using the methods disclosed herein.For example, antibodies that bind to the polypeptides of the inventioncan be isolated using the antigen microarray described elsewhere herein.

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also disclosed are antibody fragments orpolymers of those immunoglobulin molecules, and human or humanizedversions of immunoglobulin molecules or fragments thereof, as long asthey are chosen for their ability to interact with the polypeptidesdisclosed herein. “Antibody fragments” are portions of a completeantibody. A complete antibody refers to an antibody having two completelight chains and two complete heavy chains. An antibody fragment lacksall or a portion of one or more of the chains. Examples of antibodyfragments include, but are not limited to, half antibodies and fragmentsof half antibodies. A half antibody is composed of a single light chainand a single heavy chain. Half antibodies and half antibody fragmentscan be produced by reducing an antibody or antibody fragment having twolight chains and two heavy chains. Such antibody fragments are referredto as reduced antibodies. Reduced antibodies have exposed and reactivesulfhydryl groups. These sulfhydryl groups can be used as reactivechemical groups or coupling of biomolecules to the antibody fragment. Apreferred half antibody fragment is a F(ab). The hinge region of anantibody or antibody fragment is the region where the light chain endsand the heavy chain goes on.

Antibody fragments for use in antibody conjugates can bind antigens.Preferably, the antibody fragment is specific for an antigen. Anantibody or antibody fragment is specific for an antigen if it bindswith significantly greater affinity to one epitope than to otherepitopes. The antigen can be any molecule, compound, composition, orportion thereof to which an antibody fragment can bind. An analyte canbe any molecule, compound or composition of interest. For example, theantigen can be a polynucleotide of the invention. The antibodies orantibody fragments can be tested for their desired activity using the invitro assays described herein, or by analogous methods, after whichtheir in vivo therapeutic or prophylactic activities are testedaccording to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. Also disclosed are “chimeric”antibodies in which a portion of the heavy or light chain is identicalwith or homologous to corresponding sequences in antibodies derived froma particular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, as long as they exhibit the desiredantagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces monoclonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro, e.g., using the HIV Env-CD4-co-receptor complexes describedherein.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, such as an Fv,Fab, Fab′, or other antigen-binding portion of an antibody, can beaccomplished using routine techniques known in the art. For instance,digestion can be performed using papain. Examples of papain digestionare described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No.4,342,566, the contents of which are hereby incorporated by reference inits entirety for its teaching of papain digestion of antibodies toprepare monovaltent antibodies. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The fragments, whether attached to other sequences, can also includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acids residues, provided theactivity of the antibody or antibody fragment is not significantlyaltered or impaired compared to the non-modified antibody or antibodyfragment. These modifications can provide for some additional property,such as to remove/add amino acids capable of disulfide bonding, toincrease its bio-longevity, to alter its secretory characteristics, etc.In any case, the antibody or antibody fragment must possess a bioactiveproperty, such as specific binding to its cognate antigen. Functional oractive regions of the antibody or antibody fragment may be identified bymutagenesis of a specific region of the protein, followed by expressionand testing of the expressed polypeptide. Such methods are readilyapparent to a skilled practitioner in the art and can includesite-specific mutagenesis of the nucleic acid encoding the antibody orantibody fragment. (Zoller, M. J. Curr. Opin. Biotechnol. 3:348-354,1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody or a humanized antibody. Many non-human antibodies (e.g.,those derived from mice, rats, or rabbits) are naturally antigenic inhumans, and thus can give rise to undesirable immune responses whenadministered to humans. Therefore, the use of human or humanizedantibodies in the methods serves to lessen the chance that an antibodyadministered to a human will evoke an undesirable immune response.

The disclosed human antibodies can be prepared using any technique.Examples of techniques for human monoclonal antibody production includethose described by Cole et al. (Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol.,147(1):86-95, 1991). Human antibodies (and fragments thereof) can alsobe produced using phage display libraries (Hoogenboom et al., J. Mol.Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenicanimals. For example, transgenic, mutant mice that are capable ofproducing a full repertoire of human antibodies, in response toimmunization, have been described (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge. Antibodies having the desired activity areselected using Env-CD4-co-receptor complexes as described herein.

Optionally, the disclosed human antibodies can be made from memory Bcells using a method for Epstein-Barr virus transformation of human Bcells. (See, e.g., Triaggiai et al., An efficient method to make humanmonoclonal antibodies from memory B cells: potent neutralization of SARScoronavirus, Nat. Med. 2004 August; 10(8):871-5. (2004)), which isherein incorporated by reference in its entirety for its teaching of amethod to make human monoclonal antibodies from memory B cells). Inshort, memory B cells from a subject who has survived a naturalinfection are isolated and immortalized with EBV in the presence ofirradiated mononuclear cells and a CpG oligonucleotide that acts as apolyclonal activator of memory B cells. The memory B cells are culturedand analyzed for the presence of specific antibodies. EBV-B cells fromthe culture producing the antibodies of the desired specificity are thencloned by limiting dilution in the presence of irradiated mononuclearcells, with the addition of CpG 2006 to increase cloning efficiency, andcultured. After culture of the EBV-B cells, monoclonal antibodies can beisolated. Such a method offers (1) antibodies that are produced byimmortalization of memory B lymphocytes which are stable over a lifetimeand can easily be isolated from peripheral blood and (2) the antibodiesisolated from a primed natural host who has survived a naturalinfection, thus eliminating the need for immunization of experimentalanimals, which may show different susceptibility and, therefore,different immune responses.

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.). The antibodies disclosed herein can also beadministered to a subject. Nucleic acid approaches for antibody deliveryalso exist. The broadly neutralizing antibodies to the polypeptidesdisclosed herein and antibody fragments can also be administered tosubjects or subjects as a nucleic acid preparation (e.g., DNA or RNA)that encodes the antibody or antibody fragment, such that the subject'sown cells take up the nucleic acid and produce and secrete the encodedantibody or antibody fragment.

Nucleic Acid and Vectors

The invention is also directed to an isolated nucleic acid encoding anyone or more of the synthetic apolipoprotein E-mimicking peptidesdisclosed herein. For example, disclosed are isolated nucleic acidencoding the disclosed synthetic apolipoprotein E-mimicking peptides,wherein the nucleic acid comprises DNA, RNA and/or cDNA. It would beroutine for one with ordinary skill in the art to make a nucleic acidthat encodes the polypeptides disclosed herein since codons for each ofthe amino acids that make up the polypeptides are known.

The disclosed nucleic acids are made up of for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. It is understood thatfor example, when a vector is expressed in a cell that the expressedmRNA will typically be made up of A, C, G, and U. Likewise, it isunderstood that if, for example, an antisense molecule is introducedinto a cell or cell environment through for example exogenous delivery,it is advantageous that the antisense molecule be made up of nucleotideanalogs that reduce the degradation of the antisense molecule in thecellular environment.

The nucleotides of the invention can comprise one or more nucleotideanalogs or substitutions. A nucleotide analog is a nucleotide whichcontains some type of modification to the base, sugar, or phosphatemoieties. Modifications to the base moiety would include natural andsynthetic modifications of A, C, G, and T/U as well as different purineor pyrimidine bases, such as uracil-5-yl (ψ), hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N², N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Oftentime base modifications can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents, such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxy ribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀, alkyl or C₂ toC₁₀ alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)CH₃, —O(CH₂)_(n)—ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S, Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety for their teaching ofmodifications and methods related to the same.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-lkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides can be through a 3′-5′ linkageor a 2′-5′ linkage, and the linkage can contain inverted polarity suchas 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference in its entirety for their teaching ofmodifications and methods related to the same.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be, for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference in its entirety fortheir teaching of modifications and methods related to the same.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference in itsentirety for their teaching of modifications and methods related to thesame. (See also Nielsen et al., Science, 254, 1497-1500 (1991)).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Numerous United States patents teach the preparation of such conjugatesand include, but are not limited to U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference in its entirety for their teaching of modifications andmethods related to the same.

The same methods of calculating homology as described elsewhere hereinconcerning polypeptides can be obtained for nucleic acids by for examplethe algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger etal. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. MethodsEnzymol. 183:281-306, 1989 which are herein incorporated by referencefor at least material related to nucleic acid alignment.

Also, disclosed are compositions including primers and probes, which arecapable of interacting with the polynucleotide sequences disclosedherein. For example, disclosed are primers/probes capable of amplifyinga nucleic acid capable of encoding one or more of the disclosedsynthetic apolipoprotein E-mimicking peptides. The disclosed primers canused to support DNA amplification reactions. Typically the primers willbe capable of being extended in a sequence specific manner. Extension ofa primer in a sequence specific manner includes any methods wherein thesequence or composition of the nucleic acid molecule to which the primeris hybridized or otherwise associated directs or influences thecomposition or sequence of the product produced by the extension of theprimer. Extension of the primer in a sequence specific manner thereforeincludes, but is not limited to, PCR, DNA sequencing, DNA extension, DNApolymerization, RNA transcription, or reverse transcription. Techniquesand conditions that amplify the primer in a sequence specific manner arepreferred. In certain embodiments the primers are used for the DNAamplification reactions, such as PCR or direct sequencing. It isunderstood that in certain embodiments the primers can also be extendedusing non-enzymatic techniques, where for example, the nucleotides oroligonucleotides used to extend the primer are modified such that theywill chemically react to extend the primer in a sequence specificmanner. Typically the disclosed primers hybridize with thepolynucleotide sequences disclosed herein or region of thepolynucleotide sequences disclosed herein or they hybridize with thecomplement of the polynucleotide sequences disclosed herein orcomplement of a region of the polynucleotide sequences disclosed herein.

The size of the primers or probes for interaction with thepolynucleotide sequences disclosed herein in certain embodiments can beany size that supports the desired enzymatic manipulation of the primer,such as DNA amplification or the simple hybridization of the probe orprimer. A typical primer or probe would be at least 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000,3500, or 4000 nucleotides long or any length inbetween.

Also disclosed are functional nucleic acids that can interact with thedisclosed polynucleotides. Functional nucleic acids are nucleic acidmolecules that have a specific function, such as binding a targetmolecule or catalyzing a specific reaction. Functional nucleic acidmolecules can be divided into the following categories, which are notmeant to be limiting. For example, functional nucleic acids includeantisense molecules, aptamers, ribozymes, triplex forming molecules, andexternal guide sequences. The functional nucleic acid molecules can actas affectors, inhibitors, modulators, and stimulators of a specificactivity possessed by a target molecule, or the functional nucleic acidmolecules can possess a de novo activity independent of any othermolecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of polynucleotide sequencesdisclosed herein or the genomic DNA of the polynucleotide sequencesdisclosed herein or they can interact with the polypeptide encoded bythe polynucleotide sequences disclosed herein. Often functional nucleicacids are designed to interact with other nucleic acids based onsequence homology between the target molecule and the functional nucleicacid molecule. In other situations, the specific recognition between thefunctional nucleic acid molecule and the target molecule is not based onsequence homology between the functional nucleic acid molecule and thetarget molecule, but rather is based on the formation of tertiarystructure that allows specific recognition to take place.

Disclosed herein are antisense molecules that interact with thedisclosed polynucleotides. Antisense molecules are designed to interactwith a target nucleic acid molecule through either canonical ornon-canonical base pairing. The interaction of the antisense moleculeand the target molecule is designed to promote the destruction of thetarget molecule through, for example, RNAseH mediated RNA-DNA hybriddegradation. Alternatively the antisense molecule is designed tointerrupt a processing function that normally would take place on thetarget molecule, such as transcription or replication. Antisensemolecules can be designed based on the sequence of the target molecule.Numerous methods for optimization of antisense efficiency by finding themost accessible regions of the target molecule exist. Exemplary methodswould be in vitro selection experiments and DNA modification studiesusing DMS and DEPC. It is preferred that antisense molecules bind thetarget molecule with a dissociation constant (k_(d)) less than or equalto 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². A representative sample of methods andtechniques which aid in the design and use of antisense molecules can befound in the following non-limiting list of U.S. Pat. Nos. 5,135,917,5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138,5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320,5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042,6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437each of which is herein incorporated by reference in its entirety fortheir teaching of modifications and methods related to the same.

Also disclosed are aptamers that interact with the disclosedpolynucleotides. Aptamers are molecules that interact with a targetmolecule, preferably in a specific way. Typically aptamers are smallnucleic acids ranging from 15-50 bases in length that fold into definedsecondary and tertiary structures, such as stem-loops or G-quartets.Aptamers can bind small molecules, such as ATP (U.S. Pat. No. 5,631,146)and theophiline (U.S. Pat. No. 5,580,737), as well as large molecules,such as reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin(U.S. Pat. No. 5,543,293). Aptamers can bind very tightly with k_(d)sfrom the target molecule of less than 10⁻¹² M. It is preferred that theaptamers bind the target molecule with a k_(d) less than 10⁻⁶, 10⁻⁸,10⁻¹⁰, or 10⁻¹². Aptamers can bind the target molecule with a very highdegree of specificity. For example, aptamers have been isolated thathave greater than a 10,000 fold difference in binding affinities betweenthe target molecule and another molecule that differ at only a singleposition on the molecule (U.S. Pat. No. 5,543,293). It is preferred thatthe aptamer have a k_(d) with the target molecule at least 10, 100,1000, 10,000, or 100,000 fold lower than the k_(d) with a backgroundbinding molecule. It is preferred when doing the comparison for apolypeptide for example, that the background molecule be a differentpolypeptide. For example, when determining the specificity of aptamers,the background protein could be ef-1α. Representative examples of how tomake and use aptamers to bind a variety of different target moleculescan be found in the following non-limiting list of U.S. Pat. Nos.5,476,766; 5,503,978; 5,631,146; 5,731,424; 5,780,228; 5,792,613;5,795,721; 5,846,713; 5,858,660; 5,861,254; 5,864,026; 5,869,641;5,958,691; 6,001,988; 6,011,020; 6,013,443; 6,020,130; 6,028,186;6,030,776, and 6,051,698.

Also disclosed are ribozymes that interact with the disclosedpolynucleotides. Ribozymes are nucleic acid molecules that are capableof catalyzing a chemical reaction, either intramolecularly orintermolecularly. Ribozymes are thus catalytic nucleic acid. It ispreferred that the ribozymes catalyze intermolecular reactions. Thereare a number of different types of ribozymes that catalyze nuclease ornucleic acid polymerase type reactions which are based on ribozymesfound in natural systems, such as hammerhead ribozymes, (for example,but not limited to the following U.S. Pat. Nos. 5,334,711; 5,436,330;5,616,466; 5,633,133; 5,646,020; 5,652,094; 5,712,384; 5,770,715;5,856,463; 5,861,288; 5,891,683; 5,891,684; 5,985,621; 5,989,908;5,998,193; 5,998,203; WO 9858058 by Ludwig and Sproat; WO 9858057 byLudwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpinribozymes (for example, but not limited to the following U.S. Pat. Nos.5,631,115; 5,646,031; 5,683,902; 5,712,384; 5,856,188; 5,866,701;5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, butnot limited to the following U.S. Pat. Nos. 5,595,873 and 5,652,107).There are also a number of ribozymes that are not found in naturalsystems, but which have been engineered to catalyze specific reactionsde novo (for example, but not limited to the following U.S. Pat. Nos.5,580,967; 5,688,670; 5,807,718, and 5,910,408). Preferred ribozymescleave RNA or DNA substrates, and more preferably cleave RNA substrates.Ribozymes typically cleave nucleic acid substrates through recognitionand binding of the target substrate with subsequent cleavage. Thisrecognition is often based mostly on canonical or non-canonical basepair interactions. This property makes ribozymes particularly goodcandidates for target specific cleavage of nucleic acids becauserecognition of the target substrate is based on the target substratessequence. Representative examples of how to make and use ribozymes tocatalyze a variety of different reactions can be found in the followingnon-limiting list of U.S. Pat. Nos. 5,646,042; 5,693,535; 5,731,295;5,811,300; 5,837,855; 5,869,253; 5,877,021; 5,877,022; 5,972,699;5,972,704; 5,989,906, and 6,017,756.

Also disclosed are triplex forming functional nucleic acid moleculesthat interact with the disclosed polynucleotides. Triplex formingfunctional nucleic acid molecules are molecules that can interact witheither double-stranded or single-stranded nucleic acid. When triplexmolecules interact with a target region, a structure called a triplex isformed, in which there are three strands of DNA forming a complexdependant on both Watson-Crick and Hoogsteen base-pairing. Triplexmolecules are preferred because they can bind target regions with highaffinity and specificity. It is preferred that the triplex formingmolecules bind the target molecule with a k_(d) less than 10⁻⁶, 10⁻⁸,10⁻¹⁰, or 10⁻¹². Representative examples of how to make and use triplexforming molecules to bind a variety of different target molecules can befound in the following non-limiting list of U.S. Pat. Nos. 5,176,996;5,645,985; 5,650,316; 5,683,874; 5,693,773; 5,834,185; 5,869,246;5,874,566, and 5,962,426.

Also disclosed are external guide sequences that form a complex with thedisclosed polynucleotides. External guide sequences (EGSs) are moleculesthat bind a target nucleic acid molecule forming a complex, and thiscomplex is recognized by RNase P, which cleaves the target molecule.EGSs can be designed to specifically target a RNA molecule of choice.RNAse P aids in processing transfer RNA (tRNA) within a cell. BacterialRNAse P can be recruited to cleave virtually any RNA sequence by usingan EGS that causes the target RNA:EGS complex to mimic the natural tRNAsubstrate. (WO 92/03566 by Yale, and Forster and Altman, Science238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can beutilized to cleave desired targets within eukarotic cells. (Yuan et al.,Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO95/24489 by Yale; Yuan and Altman, EMBO J. 14:159-168 (1995), andCarrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).Representative examples of how to make and use EGS molecules tofacilitate cleavage of a variety of different target molecules can befound in the following non-limiting list of U.S. Pat. Nos. 5,168,053;5,624,824; 5,683,873; 5,728,521; 5,869,248, and 5,877,162.

Also disclosed are polynucleotides that contain peptide nucleic acids(PNAs) compositions. PNA is a DNA mimic in which the nucleobases areattached to a pseudopeptide backbone (Good and Nielsen, AntisenseNucleic Acid Drug Dev. 1997; 7(4) 431-37). PNA is able to be utilized ina number of methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Areview of PNA including methods of making, characteristics of, andmethods of using, is provided by Corey (Trends Biotechnol 1997 June;15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of an mRNAsequence based on the disclosed polynucleotides, and such PNAcompositions may be used to regulate, alter, decrease, or reduce thetranslation of the disclosed polynucleotides transcribed mRNA, andthereby alter the level of the disclosed polynucleotide's activity in ahost cell to which such PNA compositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science Dec. 6, 1991;254(5037):1497-500; Hanvey et al., Science. Nov. 27, 1992;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med. Chem. 1996 January;4(1):5-23). This chemistry has three important consequences: firstly, incontrast to DNA or phosphorothioate oligonucleotides, PNAs are neutralmolecules; secondly, PNAs are achirial, which avoids the need to developa stereoselective synthesis; and thirdly, PNA synthesis uses standardBoc or Fmoc protocols for solid-phase peptide synthesis, although othermethods, including a modified Merrifield method, have been used. PNAmonomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med. Chem. 1995 April; 3(4):437-45).The manual protocol lends itself to the production of chemicallymodified PNAs or the simultaneous synthesis of families of closelyrelated PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography, providing yields and purity ofproduct similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med. Chem. 1995 April; 3(4):437-45; Petersen et al., J PeptSci. 1995 May-June; 1(3):175-83; Orum et al., Biotechniques. 1995September; 19(3):472-80; Footer et al., Biochemistry. Aug. 20, 1996;35(33): 10673-9; Griffith et al., Nucleic Acids Res. Aug. 11, 1995;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. Jun. 6, 1995;92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. Mar. 14, 1995;92(6):1901-5; Gambacorti-Passerini et al., Blood. Aug. 15, 1996;88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. Nov. 11, 1997;94(23):12320-5; Seeger et al., Biotechniques. 1997 September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimericmolecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (Anal Chem. Dec. 15, 1993; 65(24):3545-9) and Jensenet al. (Biochemistry. Apr. 22, 1997; 36(16):5072-7). Rose uses capillarygel electrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology. Other applications of PNAs that have beendescribed and will be apparent to the skilled artisan include use in DNAstrand invasion, antisense inhibition, mutational analysis, enhancers oftranscription, nucleic acid purification, isolation of transcriptionallyactive genes, blocking of transcription factor binding, genome cleavage,biosensors, in situ hybridization, and the like.

Optionally, isolated polypeptides or isolated nucleotides can also bepurified, e.g., are at least about 90% pure, more preferably at leastabout 95% pure and most preferably at least about 99% pure. An“isolated” polypeptide or an “isolated” polynucleotide is one that isremoved from its original environment. For example, anaturally-occurring polypeptide or polynucleotide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem.

Also disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular polynucleotide is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the polynucleotide are discussed, specifically contemplated iseach and every combination and permutation of polynucleotide and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C—F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

It is understood that one way to define any known variants andderivatives or those that might arise, of the disclosed genes andproteins herein is through defining the variants and derivatives interms of homology to specific known sequences. Specifically disclosedare variants of the genes and proteins herein disclosed which have atleast, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percenthomology to the stated sequence. Those of skill in the art readilyunderstand how to determine the homology of two proteins or nucleicacids, such as genes. For example, the homology can be calculated afteraligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference in its entirety and at least for materialrelated to hybridization of nucleic acids). As used herein “stringenthybridization” for a DNA:DNA hybridization is about 68° C. (in aqueoussolution) in 6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein. Optionally, one or more of the isolatedpolynucleotides of the invention are attached to a solid support. Solidsupports are disclosed herein.

Also disclosed herein are arrays comprising polynucleotides capable ofspecifically hybridizing to nucleic acid capable of encoding thedisclosed synthetic apolipoprotein E mimicking peptides. Also disclosedare arrays comprising polynucleotides capable of specificallyhybridizing to nucleic acid capable of encoding the disclosed syntheticapolipoprotein E mimicking peptides.

Solid supports are solid-state substrates or supports with whichmolecules, such as analytes and analyte binding molecules, can beassociated. Analytes, such as calcifying nano-particles and proteins,can be associated with solid supports directly or indirectly. Forexample, analytes can be directly immobilized on solid supports. Analytecapture agents, such a capture compounds, can also be immobilized onsolid supports. For example, disclosed herein are antigen binding agentscapable of specifically binding to nucleic acid capable of encoding thedisclosed synthetic apolipoprotein E mimicking peptides. A preferredform of solid support is an array. Another form of solid support is anarray detector. An array detector is a solid support to which multipledifferent capture compounds or detection compounds have been coupled inan array, grid, or other organized pattern. Solid-state substrates foruse in solid supports can include any solid material to which moleculescan be coupled. This includes materials such as acrylamide, agarose,cellulose, nitrocellulose, glass, polystyrene, polyethylene vinylacetate, polypropylene, polymethacrylate, polyethylene, polyethyleneoxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon,silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, andpolyamino acids. Solid-state substrates can have any useful formincluding thin film, membrane, bottles, dishes, fibers, woven fibers,shaped polymers, particles, beads, microparticles, or a combination.Solid-state substrates and solid supports can be porous or non-porous. Apreferred form for a solid-state substrate is a microtiter dish, such asa standard 96-well type. In preferred embodiments, a multiwell glassslide can be employed that normally contain one array per well. Thisfeature allows for greater control of assay reproducibility, increasedthroughput and sample handling, and ease of automation.

Different compounds can be used together as a set. The set can be usedas a mixture of all or subsets of the compounds used separately inseparate reactions, or immobilized in an array. Compounds usedseparately or as mixtures can be physically separable through, forexample, association with or immobilization on a solid support. An arraycan include a plurality of compounds immobilized at identified orpredefined locations on the array. Each predefined location on the arraygenerally can have one type of component (that is, all the components atthat location are the same). Each location will have multiple copies ofthe component. The spatial separation of different components in thearray allows separate detection and identification of thepolynucleotides or polypeptides disclosed herein.

Although preferred, it is not required that a given array be a singleunit or structure. The set of compounds may be distributed over anynumber of solid supports. For example, at one extreme, each compound maybe immobilized in a separate reaction tube or container, or on separatebeads or microparticles. Different modes of the disclosed method can beperformed with different components (for example, different compoundsspecific for different proteins) immobilized on a solid support. Somesolid supports can have capture compounds, such as antibodies, attachedto a solid-state substrate. Such capture compounds can be specific forcalcifying nano-particles or a protein on calcifying nano-particles.Captured calcifying nano-particles or proteins can then be detected bybinding of a second, detection compound, such as an antibody. Thedetection compound can be specific for the same or a different proteinon the calcifying nano-particle.

Methods for immobilizing antibodies (and other proteins) to solid-statesubstrates are well established. Immobilization can be accomplished byattachment, for example, to aminated surfaces, carboxylated surfaces orhydroxylated surfaces using standard immobilization chemistries.Examples of attachment agents are cyanogen bromide, succinimide,aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents,epoxides and maleimides. A preferred attachment agent is theheterobifunctional cross-linker N-[γ-Maleimidobutyryloxy] succinimideester (GMBS). These and other attachment agents, as well as methods fortheir use in attachment, are described in Protein immobilization:fundamentals and applications, Richard F. Taylor, ed. (M. Dekker, NewYork, 1991); Johnstone and Thorpe, Immunochemistry In Practice(Blackwell Scientific Publications, Oxford, England, 1987) pages 209-216and 241-242, and Immobilized Affinity Ligands; Craig T. Hermanson etal., eds. (Academic Press, New York, 1992) which are incorporated byreference in their entirety for methods of attaching antibodies to asolid-state substrate. Antibodies can be attached to a substrate bychemically cross-linking a free amino group on the antibody to reactiveside groups present within the solid-state substrate. For example,antibodies may be chemically cross-linked to a substrate that containsfree amino, carboxyl, or sulfur groups using glutaraldehyde,carbodiimides, or GMBS, respectively, as cross-linker agents. In thismethod, aqueous solutions containing free antibodies are incubated withthe solid-state substrate in the presence of glutaraldehyde orcarbodiimide.

A preferred method for attaching antibodies or other proteins to asolid-state substrate is to functionalize the substrate with an amino-or thiol-silane, and then to activate the functionalized substrate witha homobifunctional cross-linker agent such as (Bis-sulfo-succinimidylsuberate (BS³) or a heterobifunctional cross-linker agent such as GMBS.For cross-linking with GMBS, glass substrates are chemicallyfunctionalized by immersing in a solution ofmercaptopropyltrimethoxysilane (1% vol/vol in 95% ethanol pH 5.5) for 1hour, rinsing in 95% ethanol and heating at 120° C. for 4 hrs.Thiol-derivatized slides are activated by immersing in a 0.5 mg/mlsolution of GMBS in 1% dimethylformamide, 99% ethanol for 1 hour at roomtemperature. Antibodies or proteins are added directly to the activatedsubstrate, which are then blocked with solutions containing agents suchas 2% bovine serum albumin, and air-dried. Other standard immobilizationchemistries are known by those of skill in the art.

Each of the components (compounds, for example) immobilized on the solidsupport preferably is located in a different predefined region of thesolid support. Each of the different predefined regions can bephysically separated from each other of the different regions. Thedistance between the different predefined regions of the solid supportcan be either fixed or variable. For example, in an array, each of thecomponents can be arranged at fixed distances from each other, whilecomponents associated with beads will not be in a fixed spatialrelationship. In particular, the use of multiple solid support units(for example, multiple beads) will result in variable distances.

Components can be associated or immobilized on a solid support at anydensity. Components preferably are immobilized to the solid support at adensity exceeding 400 different components per cubic centimeter. Arraysof components can have any number of components. For example, an arraycan have at least 1,000 different components immobilized on the solidsupport, at least 10,000 different components immobilized on the solidsupport, at least 100,000 different components immobilized on the solidsupport, or at least 1,000,000 different components immobilized on thesolid support.

Optionally, at least one address on the solid support is the sequencesor part of the sequences set forth in any of the nucleic acid sequencesdisclosed herein. Also disclosed are solid supports where at least oneaddress is the sequences or portion of sequences set forth in any of thepeptide sequences disclosed herein. Solid supports can also contain atleast one address is a variant of the sequences or part of the sequencesset forth in any of the nucleic acid sequences disclosed herein. Solidsupports can also contain at least one address is a variant of thesequences or portion of sequences set forth in any of the peptidesequences disclosed herein.

Also disclosed are antigen microarrays for multiplex characterization ofantibody responses. For example, disclosed are antigen arrays andminiaturized antigen arrays to perform large-scale multiplexcharacterization of antibody responses directed against thepolypeptides, polynucleotides and antibodies described herein, usingsubmicroliter quantities of biological samples as described in Robinsonet al., Autoantigen microarrays for multiplex characterization ofautoantibody responses, Nat. Med., 8(3):295-301 (2002), which in hereinincorporated by reference in its entirety for its teaching ofcontructing and using antigen arrays to perform large-scale multiplexcharacterization of antibody responses directed against structurallydiverse antigens, using submicroliter quantities of biological samples.

Protein variants and derivatives are well understood to those of skillin the art and can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional or deletionalvariants. Polypeptide variants generally encompassed by the presentinvention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity(determined as described below), along its length, to a polypeptidesequences set forth herein.

Also disclosed are vectors comprising isolated nucleic acids encodingthe synthetic apolipoprotein E-mimicking peptides described herein. Incertain embodiments, the invention provides a vector comprising anucleic acid encoding at least one of the peptides of the presentinvention, e.g., at least one of SEQ ID NOS: 11-14 and 18-61. Forexample, disclosed are expression vectors comprising the polynucleotidesdescribed elsewhere herein, operably linked to a control element.

Also disclosed herein are host cells transformed or transfected with anexpression vector comprising the polynucleotides described elsewhereherein. Also disclosed are host cells comprising the expression vectorsdescribed herein. For example, disclosed is a host cell comprising anexpression vector comprising the polynucleotides described elsewhereherein, operably linked to a control element. Host cells can beeukaryotic or prokaryotic cells. Also disclosed are recombinant cellscomprising isolated nucleic acids encoding the disclosed syntheticapolipoprotein E-mimicking peptides. Further disclosed are recombinantcells producing the disclosed synthetic apolipoprotein E-mimickingpeptides.

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991). Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

Expression vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)). For example, disclosed hereinare expression vectors comprising an isolated polynucleotide capable ofencoding one or more of the disclosed synthetic apolipoproteinE-mimicking peptides operably linked to a control element.

The “control elements” present in an expression vector are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and thelike may be used. In mammalian cell systems, promoters from mammaliangenes or from mammalian viruses are generally preferred. If it isnecessary to generate a cell line that contains multiple copies of thesequence encoding a polypeptide, vectors based on SV40 or EBV may beadvantageously used with an appropriate selectable marker.

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters (e.g., beta actin promoter). Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment, which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Additionally, promoters from the host cell or relatedspecies can also be used.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell. Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell. Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor or enhancer may be specifically activated either by lightor specific chemical events which trigger their function. Systems can beregulated by reagents such as tetracycline and dexamethasone. There arealso ways to enhance viral vector gene expression by exposure toirradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

Optionally, the promoter or enhancer region can act as a constitutivepromoter or enhancer to maximize expression of the polynucleotides ofthe invention. In certain constructs the promoter or enhancer region beactive in all eukaryotic cell types, even if it is only expressed in aparticular type of cell at a particular time. A preferred promoter ofthis type is the CMV promoter (650 bases). Other preferred promoters areSV40 promoters, cytomegalovirus (full length promoter), and retroviralvector LTR.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases.

The expression vectors can include a nucleic acid sequence encoding amarker product. This marker product is used to determine if the gene hasbeen delivered to the cell and once delivered is being expressed.Preferred marker genes are the E. coli lacZ gene, which encodesβ-galactosidase, and the gene encoding the green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as an isolated polynucleotide capable ofencoding one or more of the disclosed synthetic apolipoproteinE-mimicking peptides into the cell without degradation and include apromoter yielding expression of the gene in the cells into which it isdelivered. In some embodiments the isolated polynucleotides disclosedherein are derived from either a virus or a retrovirus. Viral vectorsare, for example, Adenovirus, Adeno-associated virus, Herpes virus,Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbisand other RNA viruses, including these viruses with the HIV backbone.Also preferred are any viral families which share the properties ofthese viruses which make them suitable for use as vectors. Retrovirusesinclude Murine Maloney Leukemia virus, MMLV, and retroviruses thatexpress the desirable properties of MMLV as a vector. Retroviral vectorsare able to carry a larger genetic payload, i.e., a transgene or markergene, than other viral vectors, and for this reason are a commonly usedvector. However, they are not as useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation, and cantransfect non-dividing cells. Pox viral vectors are large and haveseveral sites for inserting genes, they are thermostable and can bestored at room temperature. A preferred embodiment is a viral vectorwhich has been engineered so as to suppress the immune response of thehost organism, elicited by the viral antigens. Preferred vectors of thistype will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction abilities (i.e., ability tointroduce genes) than chemical or physical methods of introducing genesinto cells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology, Amer. Soc. forMicrobiology, pp. 229-232, Washington, (1985), which is herebyincorporated by reference in its entirity. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference in their entirety for their teaching ofmethods for using retroviral vectors for gene therapy.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serves as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. This amount of nucleicacid is sufficient for the delivery of a one to many genes depending onthe size of each transcript. It is preferable to include either positiveor negative selectable markers along with other genes in the insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell butare unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)) the teachings of which are incorporatedherein by reference in their entirety for their teaching of methods forusing retroviral vectors for gene therapy. Recombinant adenovirusesachieve gene transduction by binding to specific cell surface receptors,after which the virus is internalized by receptor-mediated endocytosis,in the same manner as wild type or replication-defective adenovirus(Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham,J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, etal., Mol. Cell. Biol., 4:1528-1533 (1984); Varga et al., J. Virology65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. Optionally, both the E1 and E3 genes are removedfrom the adenovirus genome.

Another type of viral vector that can be used to introduce thepolynucleotides of the invention into a cell is based on anadeno-associated virus (AAV). This defective parvovirus is a preferredvector because it can infect many cell types and is nonpathogenic tohumans. AAV type vectors can transport about 4 to 5 kb and wild type AAVis known to stably insert into chromosome 19. Vectors which contain thissite specific integration property are preferred. An especiallypreferred embodiment of this type of vector is the P4.1 C vectorproduced by Avigen, San Francisco, Calif., which can contain the herpessimplex virus thymidine kinase gene, HSV-tk, or a marker gene, such asthe gene encoding the green fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference in its entirety formaterial related to the AAV vector.

The inserted genes in viral and retroviral vectors usually containpromoters, or enhancers to help control the expression of the desiredgene product. A promoter is generally a sequence or sequences of DNAthat function when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors. In addition, thedisclosed polynucleotides can be delivered to a target cell in anon-nucleic acid based system. For example, the disclosedpolynucleotides can be delivered through electroporation, or throughlipofection, or through calcium phosphate precipitation. The deliverymechanism chosen will depend in part on the type of cell targeted andwhether the delivery is occurring for example in vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedexpression vectors, lipids such as liposomes, such as cationic liposomes(e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes canfurther comprise proteins to facilitate targeting a particular cell, ifdesired. Administration of a composition comprising a compound and acationic liposome can be administered to the blood, to a target organ,or inhaled into the respiratory tract to target cells of the respiratorytract. For example, a composition comprising a polynucleotide describedherein and a cationic liposome can be administered to a subjects lungcells. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell.Mol. Biol. 1:95-100 (1989); Felgner et al. Proc. Natl. Acad. Sci. USA84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the compoundcan be administered as a component of a microcapsule that can betargeted to specific cell types, such as macrophages, or where thediffusion of the compound or delivery of the compound from themicrocapsule is designed for a specific rate or dosage.

Delivery of Compositions

In the methods described herein, delivery of the compositions to cellscan be via a variety of mechanisms. As defined above, disclosed hereinare compositions comprising any one or more of the polypeptides, nucleicacids, vectors and/or antibodies described herein can be used to producea composition of the invention which may also include a carrier such asa pharmaceutically acceptable carrier. For example, disclosed arepharmaceutical compositions, comprising the synthetic apolipoproteinE-mimicking peptides disclosed herein, and a pharmaceutically acceptablecarrier

The polypeptide, nucleic acid, vector, or antibody of the invention canbe in solution or in suspension (for example, incorporated intomicroparticles, liposomes, or cells). These compositions can be targetedto a particular cell type via antibodies, receptors, or receptorligands. One of skill in the art knows how to make and use suchtargeting agents with the compositions of the invention. A targetingagent can be a vehicle such as an antibody conjugated liposomes;receptor mediated targeting of DNA through cell specific ligands, andhighly specific retroviral targeting of cells in vivo. Any such vehiclescan be part of the composition of the invention. In general, receptorsare involved in pathways of endocytosis, either constitutive or ligandinduced. These receptors cluster in clathrin-coated pits, enter the cellvia clatrhin-coated vesicles, pass through an acidified endosome inwhich the receptors are sorted, and then either recycle to the cellsurface, become stored intracellularly, or are degraded in lysosomes.The internalization pathways serve a variety of functions, such asnutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, ligand valency, and ligand concentration.

For example, the compositions described herein can comprise spharmaceutically acceptable carrier. By “pharmaceutically acceptable” ismeant a material or carrier that would be selected to minimize anydegradation of the active ingredient and to minimize any adverse sideeffects in the subject, as would be well known to one of skill in theart. Examples of carriers include dimyristoylphosphatidyl (DMPC),phosphate buffered saline or a multivesicular liposome. For example,PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in thisinvention. Other suitable pharmaceutically acceptable carriers and theirformulations are described in Remington: The Science and Practice ofPharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton,Pa. 1995. Typically, an appropriate amount ofpharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Other examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutioncan be from about 5 to about 8, or from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semi-permeablematrices of solid hydrophobic polymers containing the composition, whichmatrices are in the form of shaped articles, e.g., films, stents (whichare implanted in vessels during an angioplasty procedure), liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of compositionbeing administered. These most typically would be standard carriers foradministration of drugs to humans, including solutions such as sterilewater, saline, and buffered solutions at physiological pH.

Pharmaceutical compositions may also include carriers, thickeners,diluents, buffers, preservatives and the like, as long as the intendedactivity of the polypeptide, peptide, nucleic acid, vector of theinvention is not compromised. Pharmaceutical compositions may alsoinclude one or more active ingredients (in addition to the compositionof the invention) such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. The pharmaceutical composition may beadministered in a number of ways depending on whether local or systemictreatment is desired, and on the area to be treated.

Preparations of parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium choloride solution, Ringer'sdextrose, dextrose and sodium choloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for optical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids, or binders may be desirable. Some of the compositionsmay potentially be administered as a pharmaceutically acceptable acid-or base-addition salt, formed by reaction with inorganic acids such ashydrochloric acid, hydrobromic acid, perchloric acid, nitric acid,thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acidssuch as formic acid, acetic acid, propionic acid, glycolic acid, lacticacid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleicacid, and fumaric acid, or by reaction with an inorganic base such assodium hydroxide, ammonium hydroxide, potassium hydroxide, and organicbases such as mon-, di-, trialkyl and aryl amines and substitutedethanolamines.

Transgenic Subjects

Also disclosed are transgenic, non-human subjects comprising a nucleicacid capable of encoding one or more of the synthetic apolipoproteinE-mimicking peptides described herein. Also disclosed are transgenic,non-human subjects expressing one or more of the syntheticapolipoprotein E-mimicking peptides described herein. The subject is ananimal or a plant. The invention also provides for a transgenicnon-human subject expressing one or more of the synthetic apolipoproteinE-mimicking peptides described herein.

The animals can be produced by the process of transfecting a cell withinthe animal with any of the nucleic acid molecules disclosed herein.Methods for producing transgenic animals would be known to one of skillin the art, e.g., U.S. Pat. No. 6,201,165, to Grant, et al., issued Mar.13, 2001, entitled “Transgenic animal models for cardiac hypertrophy andmethods of use thereof.” In non-limiting embodiments, the animal is amammal, and the mammal is mouse, rat, rabbit, cow, sheep, pig, orprimate, such as a human, monkey, ape, chimpanzee, or orangutan. Theinvention also provides an animal produced by the process of adding tosuch animal (for example, during an embryonic state) any of the cellsdisclosed herein.

Compositions (such as vectors) and methods are provided, which can beused for targeted gene disruption and modification to produce thepolypeptides of the invention in any animal that can undergo genedisruption. Gene modification and gene disruption refer to the methods,techniques, and compositions that surround the selective removal oralteration of a gene or stretch of chromosome in an animal, such as amammal, in a way that propagates the modification through the germ lineof the mammal. In general, a cell is transformed with a vector, which isdesigned to homologously recombine with a region of a particularchromosome contained within the cell, as for example, described herein.This homologous recombination event can produce a chromosome which hasexogenous DNA introduced, for example in frame, with the surroundingDNA. This type of protocol allows for very specific mutations, such aspoint mutations or the insertion of DNA to encode for a new polypeptide,to be introduced into the genome contained within the cell. Methods forperforming this type of homologous recombination are known to one ofskill in the art.

Once a genetically engineered cell is produced through the methodsdescribed above, an animal can be produced from this cell through eitherstem cell technology or cloning technology. For example, if the cellinto which the nucleic acid was transfected was a stem cell for theorganism, then this cell, after transfection and culturing, can be usedto produce a transgenic organism which will contain the genemodification or disruption in germ line cells, which can then in turn beused to produce another animal that possesses the gene modification ordisruption in all of its cells. In other methods for production of ananimal containing the gene modification or disruption in all of itscells, cloning technologies can be used. These technologies are known toone of skill in the art and generally take the nucleus of thetransfected cell and either through fusion or replacement fuse thetransfected nucleus with an oocyte, which can then be manipulated toproduce an animal. The advantage of procedures that use cloning insteadof ES technology is that cells other than ES cells can be transfected.For example, a fibroblast cell, which is very easy to culture and can beused as the cell in this example, which is transfected and has a genemodification or disruption event take place, and then cells derived fromthis cell can be used to clone a whole animal. Also disclosed arenucleic acids used to modify a gene of interest that is cloned into avector designed for example, for homologous recombination.

Methods for Making the Compositions of the Invention

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted. For example, there are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. Thepeptide, polypeptides, nucleic acids and vectors of the invention can beused to make certain other aspects of the invention. For example, thepeptides and polypeptides of the invention can be used to produce theantibodies of the invention. Nucleic acids and vectors of the inventioncan be used to produce the peptides and polypeptides and otherrecombinant proteins of the invention. Host cells of the invention canbe used to make nucleic acids, proteins, peptides, antibodies, andtransgenic animals of the invention. These synthetic methods aredescribed above.

As described above, the polypeptides or peptides of the invention mayalso be used to generate antibodies, which bind specifically to thepolypeptides or fragments of the polypeptides. The resulting antibodiesmay be used in immunoaffinity chromatography procedures to isolate orpurify the polypeptide or to determine whether the polypeptide ispresent in a biological sample. In such procedures, a proteinpreparation, such as an extract, or a biological sample is contactedwith an antibody capable of specifically binding to one of thepolypeptides of the invention, sequences substantially identicalthereto, or fragments of the foregoing sequences.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or column matrix. The protein preparation isplaced in contact with the antibody under conditions under which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

The antibodies of the invention can be attached to solid supports andused to immobilize apolipoprotein E or polypeptides of the presentinvention. Polyclonal antibodies generated against the polypeptides ofthe invention can be obtained by direct injection of the polypeptidesinto an animal or by administering the polypeptides to an animal. Theantibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

C. METHODS OF USE

The invention also provides many therapeutic methods of using thenucleic acids, peptides, polypeptides, vectors, antibodies, andcompositions disclosed herein. For example, disclosed are methods forenhancing LDL binding to a cell, the method comprising contacting,mixing or associating the cell with one or more of the disclosedsynthetic apolipoprotein E-mimicking peptides. The Examples sectionbelow provides examples of how the nucleic acids, peptides,polypeptides, vectors, and antibodies, and compositions of the inventioncan be used and tested. One of skill in the art would be capable ofmodifying the methods provided in the Examples section to test and usethe nucleic acids, peptides, polypeptides, vectors, antibodies, andcompositions disclosed herein.

Also disclosed are methods for enhancing LDL binding to a cell, themethod comprising contacting, mixing or associating the cell with one ormore of the disclosed synthetic apolipoprotein E-mimicking peptideswhereby plasma LDL, plasma VLDL, or both, are affected. In addition,disclosed are methods for enhancing LDL binding to a cell, the methodcomprising contacting, mixing or associating the cell with one or moreof the disclosed synthetic apolipoprotein E-mimicking peptides wherebyplasma L(a) is affected.

Also disclosed are methods comprising administering the disclosedsynthetic apolipoprotein E-mimicking peptides to a subject, wherebyplasma LDL, plasma VLDL, or both, are affected, wherein binding of LDLto a cell of the subject is enhanced, degradation of LDL by a cell ofthe subject is increased, LDL cholesterol in the subject is lowered,binding of VLDL to a cell of the subject is enhanced, degradation ofVLDL by a cell of the subject is increased, VLDL cholesterol in thesubject is lowered, total plasma concentration of cholesterol in thesubject is lowered and/or plasma Lp(a) is lowered.

Also disclosed are methods for enhancing LDL binding to a cell, themethod comprising contacting, mixing or associating the cell with one ormore of the disclosed synthetic apolipoprotein E-mimicking peptides,thereby allowing the polypeptide to bind the LDL and enhance LDL bindingand/or uptake with the associated cell. Also provided is a method forenhancing LDL and VLDL binding to a cell in a subject, the methodcomprising administering one or more of the disclosed syntheticapolipoprotein E-mimicking peptides, or a composition thereof, to thesubject in an amount effective to increase LDL and VLDL binding to thecell of the subject. Also disclosed is a method for treating a subjectwith a “Lipid Disorder”, the method comprising administering to thesubject an effective amount of the disclosed synthetic apolipoproteinE-mimicking peptides, or a composition thereof. Also disclosed is amethod for reducing serum cholesterol in a subject, the methodcomprising administering to the subject an effective amount of thedisclosed synthetic apolipoprotein E-mimicking peptides, or acomposition thereof.

In the methods described herein, the synthetic apolipoproteinE-mimicking peptide can be administered as a composition comprising thesynthetic apolipoprotein E-mimicking peptide and a pharmaceuticallyacceptable carrier.

Administration of an effective amount of the disclosed syntheticapolipoprotein E-mimicking peptides, or a composition thereof canenhance binding of LDL to a cell, increase degradation of LDL by a cellof the subject, lower LDL cholesterol in the subject, enhance binding ofVLDL to a cell of the subject, increase degradation of VLDL by a cell ofthe subject, lower VLDL cholesterol in the subject, and/or lower totalplasma concentration of cholesterol in the subject.

Subjects for the disclosed methods can have coronary artery disease,rheumatoid arthritis, systemic lupus artherosclerosis, coronary,dysbetalipoproteinemia, and/or myocardial infarction. Subjects for thedisclosed methods can also or alternatively have inflammatory BowelDisease (IBD), systemic lupus erythematosus, Hashimoto's disease,rheumatoid arthritis, graft-versus-host disease, Sjögren's syndrome,pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome,ulcerative colitis, Crohn's disease, autoimmune hemolytic anemia,sterility, myasthenia gravis, multiple sclerosis, Basedow's disease,thrombopenia purpura, allergy; asthma, atopic disease, arteriosclerosis,myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia,and rejection after organ transplantation.

The invention also provides a method for treating a subject withcoronary artery disease or any disease or condition associated withincreased serum cholesterol. In this method, an amount of thepolypeptide of the invention, or a composition thereof, is administeredto the subject in an amount to effectively enhance cellular uptake ofserum cholesterol in the subject and thereby treat the coronary arterydisease or other associated disease in the subject. For example, theassociated disease or condition can be dysbetalipoproteinemia, highblood pressure, atherosclerosis, angina, etc. Diseases or conditionsassociated with increased serum cholesterol would be well known to oneof ordinary skill in the art.

In addition, the invention provides for a method for reducing the riskof myocardial infarction in a subject. In this method, an amount of thepolypeptide of the invention, or a composition thereof, is administeredto the subject in an amount effective to increase cellular uptake ofserum cholesterol in the subject, to thereby treat the subject andreduce risk of myocardial infarction. The invention also provides amethod for treating atherosclerosis in a subject, where an effectiveamount of the composition of the invention is administered to subject toincrease cellular uptake of serum cholesterol and to thereby treat theatherosclerosis in the subject. The invention also provides for the useof the polypeptide of the invention for the making of a composition ofthe invention, for example, to treat a disease associated with increasedserum cholesterol in a subject or to reduce LDL and/or VLDL serum levelsin a subject. The invention also provides for the use of the polypeptideof the invention for enhancing HDL function, the methods comprisingcontacting the cell with the disclosed synthetic apolipoproteinE-mimicking peptides.

The invention also provides for the use of the polypeptide of theinvention for decreasing inflammation, the methods comprising contactingthe cell with the disclosed synthetic apolipoprotein E-mimickingpeptides, wherein the peptides remove the lipid hydro-peroxides from theplasma by increasing paraoxanase. Also disclosed are methods forincreasing plasma paraoxonase (PON-1) activity, the methods comprisingcontacting the cell with the disclosed synthetic apolipoproteinE-mimicking peptides. Also disclosed are methods for inhibitingatherogenesis, the methods comprising contacting the cell with thedisclosed synthetic apolipoprotein E-mimicking peptides.

Also disclosed are methods for inhibiting atherogenesis, the methodscomprising contacting the cell with the disclosed syntheticapolipoprotein E-mimicking peptides, wherein plasma cholesterol levelsare decreased and HDL function s increased. Also disclosed are methodsfor removing atherogenic lipoproteins from vessel walls, the methodscomprising contacting the cell with the disclosed syntheticapolipoprotein E-mimicking peptides. Also disclosed are methods fordecreasing in the atherogenicity of LDL, the methods comprisingcontacting the cell with the disclosed synthetic apolipoproteinE-mimicking peptides

Numerous population and animal studies have established theatheroprotective properties of HDLs. In addition to its main atherogenicproperty of extracting cholesterol from peripheral cells andtransferring it to the liver for excretion (reverse cholesteroltransport, also referred to as RCT), HDL also poseeses anti-inflammatoryand antioxidant properties. Observations that direct infusion ofapolipoprotein A-I in animal models inhibits progression ofantiatherosclerotic plaque and, in particular, recent studies withreconstituted forms of HDL in humans demonstrating both a benefit onendothelial function and regression of atherosclerotic burden. It hasbeen shown that apoA-I mimicking peptides result in the reduction inatherosclerotic lesion formation in atherosclerosis-sentsitive mousemodels despite no change in cholesterol levels. This occurs via theformation of preβ-HDL-like particles that possess increased paroxonase-1(PON-1) activity which are able to destroy lipid hydroperoxides (LOOH)and enhance reverse cholesterol transport, the main antiatherogenicproperties described for human apoA-I.

Disclosed herein are methods comprising administering the disclosedsynthetic apolipoprotein E-mimicking peptides to a subject, wherebyplasma HDL is affected. Also disclosed herein are methods comprisingadministering the disclosed synthetic apolipoprotein E-mimickingpeptides to a subject, whereby plasma HDL function is increased. Alsodisclosed are methods comprising administering the disclosed syntheticapolipoprotein E-mimicking peptides to a subject, whereby plasma HDL isaffected, wherein the synthetic apolipoprotein E-mimicking peptide isadministered as a composition comprising the synthetic apolipoproteinE-mimicking peptide and a pharmaceutically acceptable carrier. Alsodisclosed are methods comprising administering the disclosed syntheticapolipoprotein E-mimicking peptides to a subject, whereby plasma HDL isaffected, wherein PON activity is increased, lipid hydroperoxides arecleared, atherogenic lipoproteins levels are reduced in the plasma,endothelial function is improved, and/or atherogenic lipoproteins areremoved from the vessel wall. Also disclosed are methods comprisingadministering the disclosed synthetic apolipoprotein E-mimickingpeptides to a subject, whereby plasma HDL is affected, wherein thesubject has Inflammatory Bowel Disease (IBD), systemic lupuserythematosus, Hashimoto's disease, rheumatoid arthritis,graft-versus-host disease, Sjögren's syndrome, pernicious anemia,Addison disease, scleroderma, Goodpasture's syndrome, ulcerativecolitis, Crohn's disease, autoimmune hemolytic anemia, sterility,myasthenia gravis, multiple sclerosis, Basedow's disease, thrombopeniapurpura, allergy; asthma, atopic disease, arteriosclerosis, myocarditis,cardiomyopathy, glomerular nephritis, hypoplastic anemia, and rejectionafter organ transplantation.

Also disclosed are methods for treating a subject with an “InflammatoryDisorder”, the method comprising administering to the subject aneffective amount of the disclosed synthetic apolipoprotein E-mimickingpeptides, or a composition thereof. Also disclosed are methods fortreating a subject with an “Inflammatory Disorder”, the methodscomprising administering to the subject an effective amount of thedisclosed synthetic apolipoprotein E-mimicking peptides, or acomposition thereof, wherein the synthetic apolipoprotein E-mimickingpeptide is administered as a composition comprising the syntheticapolipoprotein E-mimicking peptide and a pharmaceutically acceptablecarrier. Also disclosed are synthetic apolipoprotein E-mimickingpeptides consisting of a receptor binding domain of apolipoprotein E anda lipid-associating peptide, wherein said receptor binding domain iscovalently linked to said lipid-associating peptide in a domain switchedorientation. Subjects may be a mammal, such as a human. In anotherembodiment, the subject is an animal which can be a model system used totest human therapeutics. Non-limiting examples of such animals includedog, pig, primate, murine, feline, bovine, or equine animals.

For delivery of the nucleic acids of the invention to a cell, either invitro or in vivo, a number of direct delivery systems can be used. Theseinclude liposome fusion, gene gun injection, endocytosis,electroporation, lipofection, calcium phosphate precipitation, plasmids,viral vectors, viral nucleic acids, phage nucleic acids, phages,cosmids, or via transfer of genetic material in cells or carriers suchas cationic liposomes. Appropriate means for transfection, includingviral vectors, chemical transfectants, or physico-mechanical methodssuch as electroporation and direct diffusion of DNA, are described by,for example, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); andWolff, J. A. Nature, 352, 815-818, (1991). If ex vivo methods areemployed, cells or tissues can be removed and maintained outside thebody according to standard protocols well known in the art. Thecompositions can be introduced into the cells via any gene transfermechanism, such as, for example, calcium phosphate mediated genedelivery, electroporation, microinjection or proteoliposomes. Thetransduced cells can then be infused (e.g., in a pharmaceuticallyacceptable carrier) or homotopically transplanted back into the subjectper standard methods for the cell or tissue type. Standard methods areknown for transplantation or infusion of various cells into a subject.Such methods are well known in the art and readily adaptable for usewith the compositions and methods described herein. In certain cases,the methods will be modified to specifically function with large DNAmolecules. Further, these methods can be used to target certain diseasesand cell populations by using the targeting characteristics of thecarrier.

Therapeutic Uses

In general, when used for treatment, the therapeutic compositions may beadministered orally, parenterally (e.g., intravenously or subcutaneousadministration), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, by intracavityadministration, transdermally, or topically or the like, includingtopical intranasal administration or administration by inhalant. Thetopical administration can be ophthalmically, vaginally, rectally, orintranasally. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the disorder being treated, the particular nucleic acid orvector used, its mode of administration and the like. An appropriateamount for a particular composition and a particular subject can bedetermined by one of ordinary skill in the art using only routineexperimentation given the teachings herein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. Parenteral administration includes use of a slow release, atime release or a sustained release system such that a constant dosageis maintained.

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms of the disorder are affected. The dosage should notbe so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any counter-indications. Dosage can vary, and can be administered inone or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages forgiven classes of pharmaceutical products. For example, disclosed aremethods comprising administering one or more of the disclosed syntheticapolipoprotein E-mimicking peptides to a subject, whereby plasma LDL,plasma VLDL, or both, are affected, wherein said syntheticapolipoprotein E-mimicking peptide is administered in an amount of about0.01 mg/kg to about 5 mg/kg.

Following administration of a disclosed composition, such as a syntheticapolipoprotein E-mimicking peptide, for treating, inhibiting, orpreventing artherosclerosis, the efficacy of the therapeutic peptide canbe assessed in various ways well known to the skilled practitioner. Forinstance, one of ordinary skill in the art will understand that acomposition, such as a peptide, disclosed herein is efficacious intreating or inhibiting artherosclerosis in a subject by observing thatthe composition reduces cholesterol, LDL, or VLDL levels or reduces theamount of cholesterol present in an assay, as disclosed herein. Thecompositions that inhibit increased cholesterol levels, LDL levels, VLDLlevels artherosclerosis, or embolus formation as disclosed herein may beadministered prophylactically to patients or subjects who are at riskfor artherosclerosis, stroke, myocardial infarction, or embolusformation.

The peptides, polypeptides, nucleic acids, antibodies, vectors andtherapeutic compositions of the invention can be combined with otherwell-known therapies and prophylactic vaccines already in use. Thecompositions of the invention can be used in combination with drugs usedto stabilize the patient and limit damage to the heart. Such drugsinclude thrombolytics, aspirin, anticoagulants, painkillers andtranquilizers, beta-blockers, ace-inhibitors, nitrates,rhythm-stabilizing drugs, and diuretics. Drugs that limit damage to theheart work only if given within a few hours of the heart attack.Thrombolytic drugs that break up blood clots and enable oxygen-richblood to flow through the blocked artery increase the patient's chanceof survival if given as soon as possible after the heart attack.Thrombolytics given within a few hours after a heart attack are the mosteffective. Injected intravenously, these include anisoylated plasminogenstreptokinase activator complex (APSAC) or anistreplase, recombinanttissue-type plasminogen activator (r-tPA), and streptokinase. Thecompositions of the invention can be combined with any of these drugs.The combination of the peptides of the invention can generate anadditive or a synergistic effect with current treatments.

The peptides, polypeptides, nucleic acids, antibodies, vectors andtherapeutic compositions of the invention can also be used in thetreatment of a condition selected from the group consisting ofatherosclerotic plaque formation, atherosclerotic lesion formation,myocardial infarction, stroke, congestive heart failure, arteriolefunction, arteriolar disease, arteriolar disease associated with aging,arteriolar disease associated with Alzheimer's disease, arteriolardisease associated with chronic kidney disease, arteriolar diseaseassociated with hypertension, arteriolar disease associated withmulti-infarct dementia, arteriolar disease associated with subarachnoidhemorrhage, peripheral vascular disease, chronic obstructive pulmonarydisease (COPD), emphysema, asthma, idiopathic pulmonary fibrosis,pulmonary fibrosis, adult respiratory distress syndrome, osteoporosis,Paget's disease, coronary calcification, rheumatoid arthritis,polyarteritis nodosa, polymyalgia rheumatica, lupus erythematosus,multiple sclerosis, Wegener's granulomatosis, central nervous systemvasculitis (CNSV), Sjogren's syndrome, scleroderma, polymyositis, AIDSinflammatory response, bacterial infection, fungal infection, viralinfection, parasitic infection, influenza, avian flu, viral pneumonia,endotoxic shock syndrome, sepsis, sepsis syndrome, trauma/wound, organtransplant, transplant atherosclerosis, transplant rejection, cornealulcer, chronic/non-healing wound, ulcerative colitis, reperfusion injury(prevent and/or treat), ischemic reperfusion injury (prevent and/ortreat), spinal cord injuries (mitigating effects), cancers,myeloma/multiple myeloma, ovarian cancer, breast cancer, colon cancer,bone cancer, osteoarthritis, inflammatory bowel disease, allergicrhinitis, cachexia, diabetes, Alzheimer's disease, implanted prosthesis,biofilm formation, Crohns' disease, dermatitis, acute and chronic,eczema, psoriasis, contact dermatitis, scleroderma, Type I Diabetes,Type II Diabetes, juvenile onset diabetes, prevention of the onset ofdiabetes, diabetic nephropathy, diabetic neuropathy, diabeticretinopathy, erectile dysfunction, macular degeneration, multiplesclerosis, nephropathy, neuropathy, Parkinson's Disease, peripheralvascular disease, and meningitis.

In certain embodiments the disclosed compostions can be administered inconjunction with a drug selected from the group consisting of CETPinhibitors, FTY720, Certican, DPP4 inhibitors, Calcium channel blockers,ApoA1 derivative or mimetic or agonist, PPAR agonists, Steroids,Gleevec, Cholesterol Absorption blockers (Zetia), Vytorin, Any ReninAngiotensin pathway blockers, Angiotensin II receptor antagonist (Diovanetc), ACE inhibitors, Renin inhibitors, MR antagonist and Aldosteronesynthase inhibitor, Beta-blockers, Alpha-adrenergic antagonists, LXRagonist, FXR agonist, Scavenger Receptor B1 agonist, ABCA1 agonist,Adiponectic receptor agonist or adiponectin inducers, Stearoyl-CoADesaturase I (SCD1) inhibitor, Cholesterol synthesis inhibitors(non-statins), Diacylglycerol Acyltransferase I (DGAT1) inhibitor,Acetyl CoA Carboxylase 2 inhibitor, PAI-1 inhibitor, LP-PLA2 inhibitor,GLP-1, Glucokinase activator, CB-1 agonist, AGE inhibitor/breaker, PKCinhibitors, Anti-thrombotic/coagulants: Aspirin, ADP receptor blockers,e.g., Clopidigrel, Factor Xa inhibitor, GPIIb/IIIa inhibitor, FactorVIIa inhibitor, Warfarin, Low molecular weight heparin, Tissue factorinhibitor, Anti-inflammatory drugs: Probucol and derivative, e.g.,AGI-1067, etc., CCR2 antagonist, CX3CR1 antagonist, IL-1 antagonist,Nitrates and NO donors, and Phosphodiesterase inhibitors.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples. Rather, in view of the present disclosure thatdescribes the current best mode for practicing the invention, manymodifications and variations would present themselves to those of skillin the art without departing from the scope and spirit of thisinvention. All changes, modifications, and variations coming within themeaning and range of equivalency of the claims are to be consideredwithin their scope.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

EXAMPLES

An ideal treatment for atherosclerosis would involve rapid clearance ofplasma cholesterol and inhibition of inflammatory pathways (Navab, M.,et al., J. Lipid Res., 45:993-1007 2004; Swertfeger, D. K. et al.,Frontiers in BioSci. 6:526-535 2001). While apolipoprotein (apo) E, theprotein component of very low density lipoproteins (VLDL) is involved inthe rapid clearance of atherogenic apo B-containing lipoproteins, highdensity lipoproteins (HDL) and apolipoprotein A-I (apo A-I), the majorprotein component of HDL has been shown to exhibit anti-inflammatoryproperties. Since bringing down low density lipoprotein (LDL) levels hasyielded only approximately 30% reduction in cardiovascular risk, thenext targets against cardiovascular diseases appear to be HDL and apoA-I. Increasing HDL levels by the inhibition of cholesterol estertransfer protein appeared to increase HDL, apparently withoutimprovement in HDL function, indicating that presence of functional HDLis more important than HDL levels.

Recent advances in the apo A-I mimetic peptides indicate a possibilityto improve HDL functions (Shah, P. K. et al. Trends Cardiovasc. Med.15:291-296, 2005). This examples described below provide ways ofincorporating properties to lower plasma apo B-containing lipoprotein toapo A-I mimetic peptides, to obtain peptides with dual functions. Assuch, novel peptides that possess cationic putative receptor bindingdomain from apo E that is covalently linked to the active apo A-Imimetic peptide to yield a dual-domain peptide Ac-hE-18A-NH₂ (SEQ ID NO:12) in which residues 141-150 of apo E (LRKLRKRLLR) is linked to 18A (abaseline class A amphipathic helical peptide) were designed. Alsodesigned was a single cationic domain peptide to which the lipidhydroperoxide scavenging properties of apo A-I mimetics wereincorporated. This peptide, R18L-2Y (SEQ ID NO: 62; with the sequenceAc-GFRRFLGSWARIYRAFVG-NH₂) when folded as an α-helix, possesses Arg atthe polar face and the center of the hydrophobic face possesses aromaticresidue in n-electron cluster, capable of scavenging lipidhydroperoxides (Datta, G. et al. J. Biol. Chem. 279:26509-26517, 2004).Cationic Arg rich domains are thought to associate with ubiquitous cellsurface heparin sulfate proteoglycans (HSPG). Results show that both ofthese peptides enhance uptake of atherogenic lipoproteins in HepG2cells, clear plasma cholesterol in dyslipidemic mouse models and theyalso appear to improve HDL function. Results also show that these twocandidate peptides also inhibit atherosclerosis in apo E null mice.Previous results show that Ac-hE18A-NH₂ (SEQ ID NO: 12) dramaticallydecreases plasma cholesterol in different dyslipidemic mouse models(Datta, G. et al. Biochemistry 39:213-220 2000; Anantharamaiah, et alA-I and E. Curr. Sci. 80:11-20 2001; Datta, G. et al. J. Lipid Res.42:959-966 2001; Ramprasad, M. P. et al. J. Controlled release79:207-218 2002; Garber, D. W. et al. Atherosclerosis. 163:229-2372003), and in WHHL rabbits Garber, (D. W. et al. Atherosclerosis.163:229-237 2003).

Further results indicate that this peptide possesses anti-inflammatoryproperties. This occurs through a lowering of plasma lipid hydroperoxidelevels concomitant with a significant increase in the plasma paraoxonase(PON-1) activity. In the WHHL model, the LDL-R pathway is compromised,thus the accelerated atherogenic lipoprotein clearance is likely via thecell surface HSPG-mediated pathway, as described earlier in murinemodels (Garber, D. W. et al. Atherosclerosis. 163:229-237 2003). In asecond model of atherosclerosis, the New Zealand white (NZW) rabbits fedan atherogenic diet, a single intravenous administration (3 mg/kg) ofthe peptide significantly decreased total plasma cholesterol levels for15 days. En face analysis of the lesions after 50 days showed ˜50%lesion coverage in the saline-treated rabbits (control), while little tono lesion in the peptide-treated animals. Furthermore, in vitro studiesin HepG2 cells demonstrated that dual domain peptides specificallyincreased secretion of apo-A-I and apo E. In vitro studies have alsoshown that the dual-domain cationic peptides are recycled. The dualdomain peptides also enhance the secretion of pre-13 HDL like apoA-1-containing particles, and the effect lasts for more than 72 hrs(perhaps due to recycling dual domain cationic peptides), suggestingthat the chronic cholesterol-lowering effect of peptide in differentanimal models can be related to enhanced secretion of hepatic apoA-I inpreβ-HDL form, thus increasing the “functional HDL” levels.

Example 1 Effect of Cationic Dual-Domain Peptides on AtherogenicLipoprotein Uptake

The effect of the peptide Ac-LRKLRKRLLR-18A-NH₂ (Ac-hE18A-NH₂; SEQ IDNO: 12) in HepG2 cells and in dyslipidemic mouse models has beenpreviously described (5, 6, 7, 8, 9). These studies demonstrated thatthe peptide Ac-hE-18A-NH₂ (and not LRKLRKRLLR or Ac-18A-NH₂) associateswith atherogenic apo B-containing lipoproteins in human plasma. It wasalso shown that the peptide is able to enhance the uptake anddegradation of LDL and VLDL in HepG2 cells Datta, G. et al. Biochemistry39:213-220 2000). Preliminary results have shown that LDL-receptor wasnot involved in the clearance of plasma cholesterol. In dyslipidemicmouse models, studies showed that the peptide is able to associate withapo B48-containing lipoproteins and enhance their uptake and degradation(Datta, G. et al. Biochemistry 39:213-220 2000). In C57BL6 mice fed anatherogenic diet, apo E null mice, apo E(null)-LDL-R(null) doubleknockout mice, atherogenic lipoproteins LDL and VLDL contained mostlyapo B-48 and less of apo B-100. In experiments where atherogeniclipoprotein reduction was observed, the peptide did not reduce HDLlevels, as studied by column lipoprotein profile (CLiP) (Datta, G. etal. J. Lipid Res. 42:959-966 2001; Garber, D. W. et al. Atherosclerosis.163:229-237 2003).

Example 2 Ac-hE-18A-NH₂ Inhibits Atherosclerosis in Apo E Null Mice

Atherosclerosis inhibition studies in apo E null mice that developatherosclerosis spontaneously were also performed. Retroorbitaladministration of Ac-hE-18A-NH₂ (50 μg/mouse, 3 times weekly) for fourweeks into sixteen week old female apo E null mice showed decreasedlesion by 40% (p value<0.001) compared to the control group (n=11 incontrol and n=12 in peptide administered group). In this administrationprocedure, there was no loss of animals and no visible injury to animalswas observed, despite multiple administration (of a total of 12administrations). Lesion analysis was performed using the en facepreparations. Sixteen week old mice would have well established lesions.These results (FIG. 2) show that the peptide is able to inhibit lesionformation in apo E null mice. These results are in agreement with thepeptide being antiatherogenic. Detailed studies on the mechanism of theinhibition of atherosclerosis are described below.

Example 3

It has been shown that a portion of apo E on triglyceride-richlipoproteins, as well as on HDL is internalized and recycled (Swift, L.L. et al., J. Biol. Chem. 276:22965-22970 2001; Farkas, M. H. et al., J.Lipid Res. 45: 1546-1554 2004). Liver cells can internalize apo E whichis eventually re-released. Administration (i.v) of 100 μg of the peptideAc-hE-18A-NH₂ in to C57BL/6J mice (n=9 in each group) fed an atherogenicdiet showed a biphasic effect on plasma cholesterol levels. Initiallypeptide decreased plasma cholesterol by >65%. Lower total cholesterollevels were observed even after 8 days in the peptide administered groupcompared to the control group despite continued atherogenic dietadministration (FIG. 3). Effect on plasma cholesterol is seen even afterthe disappearance of the peptide from plasma. It is possible that theapo E-mimetic peptide is recycled. To understand the mechanism by whichthe peptide is able to exert such a dramatic effect, the effects of thepeptide on Hep G2 cells for 1) peptide bioavailability and 2) effect onHDL and apo A-I were examined. To do so, HepG2 cells were grown in MEMmedium containing 10% FCS. At 85% confluency, the cells were washed andMEM medium containing 10% LPDS was added. The cells were incubated for 5min and 60 min with 125I-labeled Ac-hE-18A-NH₂ (10 μg/ml) and with1²⁵I-labeled Ac-hE-18A-NH₂ (10 μg/ml)+LDL (10 μg/ml). At the end of theincubation time period the medium was removed and the cells washed 3times with TBA containing BSA and twice with TBA. The cells were thenincubated with buffer containing heparin at 4° C. for 1 h. The cellswere then treated with heparinase and heparitinase for 1 h at 37° C. Theheparin wash and the heparinase/heparitinase wash were counted. Thecells were aspirated in 0.1 N NaOH and counted. All the experiments weredone in triplicate and the counts expressed as a percentage of the totalcounts. FIG. 4 shows that more counts are seen in the media at 60 minafter heparinase/heparitinase wash, and correspondingly fewer counts inthe cells at 60 min. These results indicate that the peptide remainsintact on the cell surface. These results are similar to what has beenobserved for apo E, which is known to be involved in recycling (Swift,L. L. et al., J. Biol. Chem. 276:22965-22970 2001; Farkas, M. H. et al.,J. Lipid Res. 45: 1546-1554 2004).

Example 4 Inhibition of Atherosclerosis

It has been observed with an apo A-1-mimetic peptide that the peptide isable to increase HDL and apo A-I levels in mice infected with influenzavirus (Van Lenten, B. J. et al., Circulation. 106(9):1127-32, 2002). InHepG2 cells (Dashti, N. et al, J. Lipid Res. 45:1919-1928, 2004), andother mouse models it has been shown that the peptide improves theatheroprotective capacity of HDL (Anantharamaiah, G. M. et al A-I and E.Curr. Sci. 80:11-20 2001). The peptide 4F (SEQ ID NO: 17) withπ-electrons at the center of the nonpolar face, is able to form its ownparticle which can recruit apo A-I and PON and thus exertantiatherogenic effects. The peptide has also been shown to stabilizeABCA1, the membrane protein that is involved in nascent discoidal HDLsynthesis. The possible effect of Ac-hE-18A-NH₂ on HepG2 cells was alsoinvestigated. In light of previous observations with class A peptides,the effect of three peptides in the formation of HDL-like particles wasstudied. As shown in FIG. 5, compared to the supernatant from controlcells, supernatants from peptide-treated cells show a marked increase inHDL, that is smaller in size as seen by non-denaturing gradientelectrophoresis, is similar to preβ-HDL. Incubation of equal amount ofAc-hE-18A-NH₂ (SEQ ID NO: 12), Ac-hE-4F—NH₂ (SEQ ID NO: 63), and 4F (SEQID NO: 17) (on weight basis) with HepG2 cells produced preβ-HDL (FIG.5). While the amount preβ-HDL decreased with 4F in the second overnightincubation, to levels similar to that of control cell medium, even aftersecond and third overnight incubation with fresh cell medium, the othertwo cationic peptides produced significant amounts preβ HDL. Theseresults support that the dual-domain peptides perhaps due to recyclingphenominon, possess properties to secrete preβ-HDL particles much longerthan class A peptides, thus explaining the chronic antiatherogenic andanti-inflammatory effects of these peptides.

Example 5 Effect on Inflammatory Pathways

The effect of the peptide on the inflammatory response of bacteriallipopolysaccharide (LPS), a potent inducer of cytokines and celladhesion molecules was also examined. FIG. 6 shows the inhibitory effectof Ac-hE-18A-NH₂ on LPS-induced VCAM-1 expression in human umbilicalvein endothelial cells (HUVEC). Coincubation of HUVECs with LPS (1μg/ml, 6 h exposure) and Ac-hE-18A-NH₂ (50 μg/ml) showed more than 80%inhibition (lane 2, FIG. 6). As shown in FIG. 6, the present resultsshow that monocyte chemotaxis protein-1 (MCP-1) is also inhibited by thepeptide. These results indicate that the antiinflammatory properties ofthe peptide can be due to either its effect directly on LPS, or thenewly secreted apoA-I may be causing the inhibition of LPS effect onHUVECs levels or improved HDL function or both in vivo.

Example 6 Ac-hE-18A-NH₂ Enhances the Secretion of De Novo SynthesizedApo E by Macrophages

THP-1 monocyte derived macrophages were metabolically labeled with³⁵S-methionine in RPMI medium containing FBS. Macrophages (10⁶ cells)were treated with the dual-domain peptide (25 tjg/10⁶ cells) for 5.Conditioned medium was collected and cells were washed with cold PBS.Preparative cocktail containing MEM, plus lupeptin (50 tjg/ml),pepstatin A (50 tjg/ml), and aprotinin (100 kallikrein inactivatingunits/ml) were added to the medium to preserve oxidative and proteolyticdamage. The medium from control cells and peptide-treated medium wereconcentrated to equal volume and loaded quantitatively onSDS-polyacrylamide gels (4 to 20% PAGE for 2.5 h at 4° C. at 125 volts).The gel was exposed to x-ray film for overnight. Band obtained in thepeptide treated cell medium clearly had a band at 36 kDa and theintensity of this band was 4 times more than the band obtained from themedium of control cells, as determined by the densitometry (FIG. 7).Increased de novo synthesis of apo E can enhance the uptake ofatherogenic lipoproteins. In addition, apo E has anti-inflammatoryproperties and properties to enhance cholesterol efflux frommacrophages. These properties would prevent macrophages from becomingfoam cells. These studies showed that the peptide is turned over veryrapidly in vivo and maximum counts in the liver were observed. Thus thepeptide would recycle and presence of the peptide would have lastingeffect on the production of preβ HDL, increase in the synthesis of denovo apo E, and the peptide would enhance the clearance of atherogeniclipoproteins both directly (perhaps via the HSPG pathway) and indirectlyvia the increased synthesis of apo E. As presented in FIG. 8, thecationic single domain peptide R18L-2Y (SEQ ID NO: 62) (even as apeptide containing L-amino acids) inhibited atherosclerosis in apo Enull mice when orally administered.

In addition to this, it was shown that the peptide is able to stimulatethe synthesis of additional antiatherogenic proteins involved inlipoprotein metabolism (FIG. 9). THP-1 derived macrophages wereincubated with Ac-hE18A-NH₂ for 5 h and overnight (O/N). RNA wasextracted from the cells by Trizol (Invitrogen). mRNA levels weredetermined by real time PCR using SYBR green and appropriate primers forthe genes. Results were normalized against GAPDH and expressed as foldincrease over control cells (without peptide). These results show thatthe peptide Ac-hE-18A-NH₂ exerts a long-term effect that results in thedecrease of not only circulating atherogenic apo B-containinglipoproteins but also exhibits additional effects on shutting down thepro-atherogenic protein levels and increasing the levels of proteinsthat may be involved in clearing atherogenic lipoproteins. Thus, theresults can be explained by the multiple antiatherogenic andanti-inflammatory effects of this peptide.

Example 7

Although Ac-hE-18A-NH₂ enhanced the hepatic uptake and degradation ofatherogenic lipoproteins in apo E null mice, dual knockout mice(LDL-R(null)-apo E(null)), and C57BL/6 on an atherogenic diet, thepeptide had no effect on the plasma cholesterol levels of C57BL/6 onnormal chow, LDL-R(null) on normal chow or on a Western diet. Furtherinvestigations showed that n these mouse models (LDL-R (null) and C57BL6on normal chow), the peptide is not able to associate withB-100-containing particles. However, the peptide is able to associatewith human LDL (containing apo B-100) and VLDL and is able to enhanceuptake and degradation of atherogenic human lipoproteins in HepG2 cellsand in LDL-R (null) mouse model (Garber, D. W. et al. Atherosclerosis.163:229-237 2003). The reason for the difference in the properties ofapo B-100-containing human LDL and mouse LDL is not clear. Thedifference appears to be in the lipid packing between human LDL andmouse LDL that possess apo B-100. Apo B-100-containing mouse LDL doesnot allow the binding of the peptide to its surface despite the factthat the peptide possesses exceptionally high exclusion pressure value(48 dynes/cm) (Garber, D. W. et al. Atherosclerosis. 163:229-237 2003).

These observations led to the study summarized in FIG. 9. The peptideAc-hE-18A-NH₂ is able to associate with atherogenic lipoproteins fromWHHL rabbits and NZW rabbits on atherogenic diet and enhance theiruptake and degradation. Present observations in rabbits show that thepeptide is able to improve HDL function and also endothelial function.Endothelial function is closely related to the HDL function. Since HDLfunction is correlated to CETP function, rabbits are a better model forstudying the scheme shown in FIG. 10. Although CETP expressing mousemodel is available, for studying the effect of the peptide, these micehave to be crossed with atherosclerosis-sensitive mouse model(especially on an human apo A-1-expressing mouse model), which by itselfwould be a separate research project and even then the Gene Foldincrease lesions produced in these models differ significantly from thetypes of human lesions. Since the WHHL rabbit models selected here areclose to familial hypercholesterolemia in humans, and dyslipidemia canbe produced using different types of diets with varying pathology, theeffect of the peptide in two rabbit models was studied. Furthermore,similar to humans rabbits possess CETP which plays an important role inthe cholesterol metabolism. Thus, results obtained using the two rabbitsdescribed here have a direct relevance to the human atheroscleroticdisease

Example 8 Effect of the Peptide Administration in WHHL Rabbits

It has been previously demonstrated that a single administration of thepeptide Ac-hE-18A-NH₂ exerts a dramatic effect on endothelial functionand decrease in plasma cholesterol while the control peptides wereinactive (Circ. 2005; 111:3112-3118). The peptide associates with LDLfrom WHHL rabbits, modifies the LDL surface charge and removes lipidhydroperoxides (seeding molecules). Since the peptide did not associatewith the plasma LDL from LDL-R (null) mice, a study was developed todetermine if the peptide is able to associate with plasma LDL from WHHLrabbits, a model for human hyperlipoproteinemia. 100 μg of the¹²⁵I-labelled peptide was mixed with 1 ml of plasma from 6 month oldWHHL rabbit. After incubation for 1 h at room temperature, the plasmawas subjected to CLiP analysis (66). Radioactivity in differentfractions was determined and plotted on the CLiP profile. The resultsshowed that the peptide associates with LDL, the major class oflipoprotein present in WHHL plasma. The peptide-treated WHHL plasma LDLcontains reduced amounts of LOOH compared to plasma from untreated WHHLrabbits. A single bolus (15 mg/kg intravenous) administration ofAc-hE-18A-NH₂ not only reduced plasma cholesterol levels from 562±29mg/dl to 287±22 mg/dl at 18 h, in WHHL rabbits but also significantlyimproved arterial endothelial function. This improvement was associatedwith a reduction in 2 markers of oxidative stress. First, the plasmalipid hydroperoxide content was reduced significantly, an effectassociated with a 5-fold increase in HDL paraoxonase activity. Second,the formation of superoxide anion, a scavenger of nitric oxide, was alsosignificantly reduced in arteries of these animals

Because dyslipidemia and endothelial dysfunction are common features ofthe atherosclerotic disease process, these unique peptides have idealcomposite properties that ameliorate atherosclerosis. With the report onthe apoA-IMilanoli^(p)id complex infusion studies in humans (Nissen, S.E., et al. JAMA 290:2292-2300 2003), interest in HDL-based therapy hasincreased. Although the results described for apo A-IMilano aresignificant, due to the amount of protein:lipid complex to be infused(40 mg/kg of protein alone plus phospholipids), the cost of such atreatment is enormous. In this context, the present results show that asingle administration of an amphipathic helical peptide is effective indramatically reducing plasma cholesterol levels and improvingendothelial function. Large amounts of peptide can be produced andpeptide can be administered without lipid to achieve key contributoryfactors to antiatherogenic effects in vivo.

Effect of Peptide Administration to NZWrabbits on 1% Cholesterol Diet

The above results indicate that the peptide exerts an effect onatherogenic LDL in enhancing hepatic clearance and also in improving HDLfunction. It has been shown that very small amounts of D-4F, a class Aamphipathic helical peptide, modifies several HDL properties (Navab, M.,et al. Circulation 109:3215-3220 2004). D-4F reorganizes HDL to produce“pre-βHDL like” particles that are highly effective in destroying lipidhydroperoxides and thereby enhancing reverse cholesterol transport. NZWrabbits have been studied for hypercholesterolemia and relative LDL andβ-VLDL production using diets containing different amounts ofcholesterol (Holvoet, P. et al. Arterioscl. Thromb. Vasc. Biol.17:2376-2382 1997). Thus, with 0.125% (w/w) cholesterol diet, LDLcholesterol levels increase; with 0.5% and higher cholesterol levels inthe diet, □-VLDL (containing apo B-100) increases dramatically. Theseβ-VLDL particles contain increased amounts of oxidized lipids, thusenhancing the progression of atherosclerosis (Holvoet, P. et al.Arterioscl. Thromb. Vasc. Biol. 17:2376-2382 1997). To assess the effectof the peptide in this rabbit model, a 1% cholesterol diet fed NZWrabbits were utilized.

Rabbits responding to high cholesterol diet were randomized one weekafter the start of the diet to select rabbits with similar response(similar amounts of total plasma cholesterol). Ac-hE-18A-NH₂ (3 mg/kg)was administered intravenously (i.v.) 15 days after the initiation ofthe diet and rabbits were continued on high fat diet for the entirestudy period. After 14 days from the first administration, plasmasamples were taken from both the peptide-administered and salineadministered (control) rabbits (n=3 in each group). The plasma samplesfrom the peptide administered rabbits were not turbid, whereas theplasma samples from control rabbits were turbid. Significantly decreasedamounts of VLDL and LDL were also obvious. The column lipoproteinprofiles of representative rabbits from peptide administered group andcontrol show that the atherogenic lipoproteins levels decreased. Asecond dose of peptide was administered 15 days after the firsttreatment. Since the cholesterol levels remained low two weeks after thesecond administration in peptide-administered rabbits, these and salineadministered rabbits were sacrificed 51 days after the initiation of thediet. Aorta from the peptide administered and control rabbits werestained with Oil Red O. Aorta from the peptide administered rabbits had40-50% less lesion than the control rabbit aorta.

To see the cumulative effect of the peptide at a shorter interval, aslightly different protocol was utilized. Rabbits with similar levels ofplasma cholesterol upon 1% cholesterol diet administration for one weekwere selected. Peptide (7.5 mg/kg) was i.v. administered in twointervals (first one week after high fat diet initiation and the seconda week after the first peptide administration). Plasma cholesterollevels were determined at the time of administration of the peptide,before second administration and a week after second administration.Results demonstrate that in peptide-treated rabbits the plasmacholesterol was 50% less than in the control rabbits at the end of theexperiments (FIG. 11). The effect of the peptide on plasma cholesterollevels are observed even after the disappearance of the peptide fromcirculation (see FIG. 11). Using 3 mg/kg of radiolabelled peptide,turnover studies showed that the plasma clearance of the peptide (FIG.12) is much faster than that observed in WHHL rabbits. T his suggeststhat the plasma cholesterol lowering continues even after the peptidehas disappeared from the plasma compartment.

Therefore, possible reasons for the clearance of atherogeniclipoproteins can be in addition to rapid hepatic clearance similar tothe properties of apo E, modulation of HDL properties or synthesis ofmacrophage apo E. If this is true, this peptide may also exert itseffect on endothelial function. Indeed it was observed that there is arecovery of endothelial function as studied by the acetylcholinedose-dependent aortal relaxation (FIG. 13). While the control rabbits(with cholesterol levels 2000 mg/dl) after 51 days of the 1% cholesteroldiet administration have lost endothelial function completely, theaortal rings from peptide-administered rabbits show vascular responsealmost similar to aortas obtained from rabbits on a normal diet (FIG.12). These results indicate that the peptide can act by inhibitingsuperoxide anion production or by a presently unknown mechanism. It ispossible that lipid lowering can cause reduction of oxidative stress andthus inhibition of endothelial activation.

Example 9 The Concept of Single Domain Cationic Peptides, In Vitro andIn Vivo Studies

Dual domain peptide that has LRKLRKRLLR (SEQ ID NO: 1), a sequence fromapo E putative receptor binding domain, covalently linked to 18A (SEQ IDNO: 11) enhances uptake and degradation of apo B-containinglipoproteins. It has been previously shown that a synthetic model lyticpeptide (18L, FIG. 14) in the past that is able to lyse red cells(Aikawa, M., et al. Circulation 106:1390-1396 2002). It has also beenpreviously shown that that if the Lys residues are replaced by Arg,compared to the Lys-containing peptide, the resulting Arg-containingpeptide has only 2% of lysis (Aikawa, M., et al. Circulation106:1390-1396 2002). Based on the idea that central aromatic residuecluster at the center of the nonpolar face is able to scavenge lipidhydroperoxides and thus the resulting peptide is able to exhibitanti-inflammatory properties, the original R18L was modified (FIG. 15).Rearrangement of the nonpolar face of 18L to incorporate aromaticresidues at the center of the nonpolar face yields 18L-2. Addition ofTyr (for radiolabeling) yields 18L-2Y (FIG. 15). All of the Lys residueschanged to Arg results in R18L2Y (FIG. 15). The cholesterol reducingproperties of 18L-2Y and R18L-2Y were compared in apo E null mice andthese results are shown in FIG. 16. The Arg-containing peptide R18L-2Ypossessed increased ability to clear plasma cholesterol compared to18L-2Y (FIG. 16).

It has also been observed that a class A peptide (D-4F) orallyadministered, inhibits atherosclerosis in apo E null mice, even thoughthe bioavailability of the peptide is only in nanomolar quantities. Thistakes place in the absence of change in the plasma cholesterol levels.Since the π-electron density cluster has been incorporated to thisR18L-2Y peptide and the peptide is able to associate with cell surfaceproteoglycans due to the presence of positively charged Arg residues onthe polar face, even across the gut, and enters plasma, a decrease inplasma cholesterol levels was observed. As shown in FIG. 17, oraladministration of R18L-2Y (mixed with chow and administered as describedin the FIG. 17), showed significant decrease in cholesterol levels at 15days and 30 days. Based on these results, the peptides 18L-2Y andR18L-2Y were mixed with normal chow (1 mg of the peptide for 4 g ofchow) and fed to four week old female apo E null mice, ad libidum. Thestudy was continued for 6 weeks. At the end of the study period, animalswere euthanized and aortic sinus from each animal was analyzed foratherosclerotic plaque development using Oil Red O. As shown in FIG. 8,R18L-2Y treated group (and not the other two groups) had significantlyless lesion formation compared to both control mice and 18L-2Y treatedmice. As shown in FIG. 18, plasma cholesterol was also significantlyreduced in R-18L-2Y group and not in other two groups.

Example 10

Described below are studies of two major pathways for inhibitingatherogenesis, decreasing plasma cholesterol levels and improvingendothelial cell function due to changes in lipoproteins, especially theHDL function. This example centers on examples of the peptides describedabove, specifically, Ac-hE-18A-NH₂ (SEQ ID NO: 12), Ac-hE-4F—NH₂ (SEQ IDNO: 63), and R18L-2Y (SEQ ID NO: 62) as agents that are able to modulatedual properties in vivo, the rapid hepatic clearance of atherogeniclipoproteins and alteration of endothelial function. The overall designis described diagrammatically in FIG. 10. The schematic illustrates thatupon cationic peptide interaction with plasma lipoproteins severalchanges occur. (1) Peptides interact with apo B-containing atherogeniclipoprotein particles to incorporate positively charged domains. Thiswill then be recognized by the receptors on the hepatic cell surface toclear these atherogenic lipoproteins from circulation, thus inhibitingatherosclerosis. (2) Peptides modify HDL in the plasma to increase PONactivity and decrease lipid hydroperoxides (LOOH) levels (Navab, M., etal. Circulation 109:3215-3220 2004); lower plasma LOOH levels lead toincreased functional nitrous oxide (NO) levels and restoration ofendothelial function in dyslipidemic animal models. Inhibition ofmonocyte chemotactic protein-1 (MCP-1) synthesis can then result inreduced monocyte chemotaxis and macrophage accumulation; thus resultingin inhibition of atherosclerosis.

Uptake of Apo B-Containing Lipoproteins in Hep G2 Cells, Mouse andRabbit Hepatocytes

Results observed in HepG2 cells indicate that peptide associates withapo B-containing plasma lipoproteins to incorporate positive charges onthe lipoprotein surface. This enables the apo B-containing lipoproteinsto interact with heparan sulphate proteoglycans (HSPGs). The effect ofthese peptides on the mode of reduction of plasma cholesterol levels inmouse models and the two rabbit models can be determined. In NZW rabbitsfed a 1% cholesterol-diet, a reduction of plasma cholesterol is observedwhich lasts for 14 days after the peptide administration; whereas in theWHHL rabbits, the reduction is initially rapid and returns to originallevels within 3 days. Since the WHHL rabbit model is LDL-receptordefective, the differential effects of the peptide in two models can bedue to differences in the receptor-mediated clearance pathways ofatherogenic lipoproteins. HepG2 cells, primary hepatocytes from apo Enull and LDL-R null mice, and primary rabbit hepatocytes can be used todetermine the molecular factors in the receptor-mediated clearancepathways of atherogenic lipoproteins.

Isolated hepatocytes can be isolated from two mouse models withpeptide:apo B-containing lipoprotein complexes (to determine possibleeffect of the peptides on cell surface lipoprotein receptors).Initially, human plasma lipoproteins can be used to determine the extentof internalization in hepatocytes from different animal models. Thesestudies can determine the commonality and differences in hepatocytes andthe ability of the peptides to modify lipoprotein surfaces. Thesemodifications can be correlated to the uptake and degradation bydifferent hepatocytes and in presence of peptides R18L-2Y, Ac-hE-18A-NH₂and Ac-hE-4F—NH₂. The role of LDL-R and LRP receptors in the uptake anddegradation of these complexes can also be determined. Whether thepeptides enhance the uptake and degradation of apo B-containinglipoproteins via the HSPG-mediated pathway using heparinase/heparatinasecan be determined as described by Datta et al. (Datta, G. et al.Biochemistry 39:213-220 2000) as well as if and by what mechanism(s)peptide-lipoprotein complexes are internalized. Using mutant CHO-cellsthat lack proteolysis (Esko, J. D. et al. Curr. Opin. Biol.: 3:805-8161991) the role of HSPG in the uptake and degradation can also bedetermined. The role of LRP can be studied using LRP-deficientfibroblasts. These procedures are described by Datta et al. using humanplasma LDL and VLDL samples (Datta, G. et al. Biochemistry 39:213-2202000; Datta, G. et al. J. Lipid Res. 42:959-966 2001). The effect ofpeptide administration on the receptor-associating ability of apoB-containing lipoproteins isolated from mice administered with thepeptide (and blood sampled at earlier and later time points, within 30min and 4 h, respectively, after peptide administration) will be studiedand compared to apo B-containing lipoproteins from control mice. Thisrequires a careful characterization of the lipoprotein properties toidentify potential changes in receptor-ligand interactions as well asoxidation status. Controls for these experiments are normal cell linesthat possess receptors.

In rabbits, apo A-I is synthesized in the intestine and not in the liver(Pan, T. C., et al. Eur. J. Biochem. 30:99-104 1987). Thus, thesestudies can determine if de novo synthesis of apo E and possiblemechanisms of uptake of atherogenic apo B-containing lipoproteins. Inrabbit hepatocyte studies, lipoproteins isolated frompeptide-administered WHHL rabbits and NZW rabbits on high fat diet canbe used for these studies. In WHHL rabbits, due to receptor defect,normal receptor-mediated atherogenic binding and uptake is compromised.Thus, any uptake of atherogenic lipoproteins is due to HSPG and/or LRPpathway. Treatment with peptides can reduce or even eliminate LOOH fromthe surface of lipoproteins. These lipoproteins can then be studied forreceptor-mediated binding and uptake in HepG2 cells and in primaryculture from rabbit hepatocytes. To determine if removal or reduction ofLOOH alone can modify hepatic uptake of these atherogenic lipoproteins,a peptide that does not incorporate positive charges on the lipoproteinsurface but yet is capable of reducing LOOH levels can be utilized. Sucha peptide is 4F (SEQ ID NO: 17) or other class A peptides in this series(Navab, M., et al. Circulation 109:3215-3220 2004). This study will beable to distinguish between positively charged peptide incorporationenhancing the uptake of atherogenic lipoproteins versus removal orreduction of LOOH from lipoprotein surface. These studies can thusdetermine whether the class A part or the positively charged apo E part(for example, LRKLRKRLLR; SEQ ID NO: 1) or a combination of the two isresponsible for the enhanced hepatic uptake. In the single domainpeptide R18L-2Y, these studies can provide information on the make up ofthe nonpolar face for reducing plasma LOOH levels since 4F serves as acontrol peptide for determining the difference between cationic natureversus class A motif on their biological properties. Both 4F and R18L-2Ypossess clustered π-electrons at the center of the nonpolar face.Observations of the peptides indicate that by covalently linking the twodomains, a novel new peptide whose properties are not just the sum ofthe properties of two domains but a peptide with unique properties havebeen identified. Use of hepatocytes from apo E null and LDL-R null micecan also provide information on the role of LDL-receptor, HSPG and/orLRP pathway for enhanced atherogenic lipoprotein uptake. These twosystems can provide information on how much of the effect is due to thedirect effect of the peptide versus the enhanced synthesis of endogenousapo E, since apo E will not be synthesized in apo E null mousehepatocytes. These investigations will complement established cell linestudies. Use of rabbit hepatocytes will give information on the hepaticuptake of atherogenic particles in rabbits. As such, hepatocytes fromWHHL rabbits and NZW rabbits can be used to understand thepeptide-mediated uptake. It is possible that the single domain peptideR18L-2Y would inhibit atherosclerosis and decrease apo B-containinglipoproteins in an entirely different mechanism. Using ¹⁴C-radiolabeledpeptide we will determine the ability of each peptide to recycle andpossess chronic antiatherogenic properties either via the synthesis ofpreβ-HDL or increased synthesis of antiatherogenic proteins such as apoE, apo A-I and possible receptors, as shown in FIGS. 5, 7 and 9.

To determine if peptides alter the synthesis of antiatherogenicproteins, Hep G2 cells and hepatocytes obtained from these animal modelscan be incubated with peptides and levels of proteins and mRNA levelscan be determined using suitable primers for these proteins. Results onthese lines are provided in FIG. 9. Based on the differences seen in twomouse (apo E null and LDL-R null mouse models) and rabbit models,results on the induction of apo A-I synthesis in the preβ-DL form inHepG2 cells (FIG. 5) (Dashti, N. et al, J. Lipid Res. 45:1919-19282004), the synthesis of one or more of the following proteins (a) apo E,(b) LDL-R, (c) apo A-I, (d) chylomicron-remnant receptor, (e) LRP, (f)LPL, (g) VLDL-R can be studied. If peptides alter the properties oflipoproteins, there can be no changes in the levels of proteins or mRNA.These studies can separate the direct and indirect antiatherogeniceffects of peptides. Use of hepatocytes from these models can alsodetermine the possible differences in the mechanism of action of thesepeptides in these two animal models.

Hepatic clearance of atherogenic lipoproteins is consideredantiatherogenic; however, macrophage uptake is atherogenic. Apo E hasbeen shown to mediate hepatic uptake of atherogenic lipoproteins(Mahley, R. W. Science. 240:622-630 1988). Macrophages secrete LPL intothe culture medium. Several factors, such as cytokines (interleukins) inthe artery wall, can regulate macrophage LPL expression. Inhibition ofmacrophage LPL activity by apo E has been thought to inhibit uptake oflipoprotein remnants by macrophages but divert them to apo E-mediatedhepatic uptake. Zilversmit and Witztum and co-workers have suggestedthat LPL present on the endothelial surface may produce remnantlipoproteins which may be potentially atherogenic (Zilversmit, D. E.,Circulation 60:473-485 (1979); (Yla-Herttuala, S. et al. Proc. Natl.Acad. Sci. U.S.A. 88:10143-10147 1991). With this in mind, the role ofpeptide in vitro in modulating LPL activity can be determined.Previously, it has been demonstrated in vitro that class A peptidesmodulate LPL activity (Chung, B. H., et al. J. Lipid Res. 37:1099-11121996). One of the major preliminary findings in both the rabbit modelsis that the peptide(s)-mediates accelerated clearance of remnantlipoproteins, and VLDL. Plasma from peptide-administered rabbits showsno turbidity whereas plasma from rabbits not treated with the peptideshows turbidity. This result is corroborated by the results in FIGS. 11and 12) which demonstrate that VLDL-like particles are significantlyreduced in NZW (on 1% cholesterol diet and peptide administered) andWHHL rabbits, which show a significant decrease in plasma TG levels (inWHHL). The total plasma cholesterol levels do not increase in thepeptide-treated rabbits despite continued feeding of the highcholesterol diet. However, the plasma residence time for the peptide isrelatively short (t_(1/2)=1 to 2 min) as shown in FIG. 9. It can bedetermined whether the peptide blocks accumulation of VLDL, TGRLP,modified-LDL (containing increased LOOH levels such as plasma from WHHLrabbits). The levels of mRNA and protein can also be determined in thesame studies.

Whether the peptide analogs exert their effect by inhibiting uptake ofapo B-containing lipoproteins by monocyte-macrophages and/or if theypromote efflux of cholesterol from the cholesterol loaded macrophage canalso be determined. Previously published results indicate that class Aamphipathic helical peptides inhibit the ability of VLDL-induced foamcell formation in cultured THP-1 monocyte derived macrophages (Chung, B.H., et al. J. Lipid Res. 37:1099-1112 1996). The procedure fordetermining LPL activity modulation and effect on THP-1 monocyte-derivedaccumulation has been described in detail in Chung et al (Chung, B. H.,et al. J. Lipid Res. 37:1099-1112 1996). and the described studies canbe used to determine the effects of the present peptides. As such, VLDLisolated from these two rabbit models (with and without peptideadministration) can be incubated with isolated LPL and determine theamount of free fatty acids obtained as an indication of differences inLPL activity. If peptides bind to HSPG (similar to what is proposed, forLPL) atherogenic lipoproteins can then bind and get internalized viaLDL-receptor related protein as suggested previously (Besiegel, U. etal. Proc. Natl. Acad. Sci. U.S.A. 88:8342-8346 1991). These studies canbe performed using both the single domain and dual-domain peptides.

Results also indicate apo B-48-enriched 31-VLDL appears in plasma ofcholesterol-fed rabbits. The inhibition of atherosclerosis due to thepeptides can be due to masking apo B domains involved in high affinityuptake of these lipoproteins by the TGRLP/apo B-48 receptor. A domain ofapo B-48 has been shown to be sufficient for high affinity binding ofTGRLP/apo B-48 receptor (Brown M. L., et al. Proc. Natl. Acad. Sci. USA97:7488-7493 (2000). With this in mind, apo B-48 receptor transfectedCHO cells incubated at 37° C. for 3 h with chylomicron Sf>400 with apoB48 as the only apo B48 species at 100 tjg TG/ml RPMI with and withoutthe peptide in a concentration dependent manner (5 tjg to 100 jtg) andthen stained with Oil Red O to detect cytoplasmic neutral lipid dropletscan be performed. Vector only transfected cells can be incubated withchylomicrons under identical conditions and stained with Oil Red O.

Regarding whether the peptides reduce the atherogenic properties of LDL(i.e., effect on monocyte chemotaxis and enhance hepatic receptorbinding properties in vitro) both in vitro and in vivo results indicatethat the dual domain peptide changes HDL properties. Using the methodsdescribed by Dashti et al. (Dashti, N. et al, J. Lipid Res. 45:1919-19282004), it can be determined if there is increase in the synthesis of apoA-I and the possible mechanism. In previous studies peptideAc-hE-18A-NH₂ resulted in increased PON activity in HDL, which destroyslipid hydroperoxides. Whether these changes alter levels of monocytechemotactic protein and adhesion molecules such as VCAM-1 can also bedetermined (FIG. 6). Preliminary studies indicate that these peptideswould possess much greater efficiency in reducing atherogenic propertiesof LDL.

Using lipoproteins isolated from the peptide-administered and controlrabbits, the extent of LDL (or VLDL)-mediated monocyte chemotaxis can bedetermined using the endothelial cells-smooth muscle cells coculturesystem as described in (Navab, M., et al., J. Lipid Res. 41:1495-15082000; (Navab, M., et al., Circulation. 105: 290-302 2002). Usingcultured hepatocytes, whether the presence of peptides enhances theuptake of atherogenic lipoproteins from rabbits treated with peptidesand control rabbits can also be determined.

Example 11 Changes in Apo A-I and Apo E-Containing Particles and theirAnti-Inflammatory Properties

Changes in apo A-I and apo E-containing particles and theiranti-inflammatory properties can be determined by analyzing cellsupernatants for the levels of different apolipoproteins by SDS gradientgels and scanning the bands for quantitation after Western blotting fordifferent apolipoproteins. As described above, the changes in thelipoproteins secreted using different peptides can be determined.Production of pre-31 HDL is correlated to increased beneficial effectsof HDL subpopulation in terms of clearance of lipid hydroperoxides fromapo B-containing lipoprotein surfaces. These are related to inhibitionof LDL-induced monocyte chemotaxis. Reduction in levels of oxidized LDLhas been shown to inhibit cytokine and adhesion molecules production. Asdiscussed above, whether the mRNA levels are correlated to increasedlevels of apolipoproteins can be determined. These studies candistinguish between the increase in the protein synthesis due to effecton mRNA levels vs being simply due to increased secretion and (asopposed to degradation) due to increased phospholipid levels as shown byus in published results (Dashti, N. et al, J. Lipid Res. 45:1919-19282004). The methods published by Dashti et al. (Dashti, N. et al, J.Lipid Res. 45:1919-1928 2004) can be used to determine the effect ofdifferent peptides on possible changes in the levels of apolipoproteinsA-I and E, increased levels of which have been shown to beantiatherogenic. Cells labeled with ³⁵S-Methionine to follow the newprotein synthesis, can be used as described Dashti et al. (Dashti, N. etal, J. Lipid Res. 45:1919-1928 2004). ³H-glycerol can be used todetermine changes in the lipid composition upon peptide incubation.HepG2 can be incubated in serum free MEM and incorporation of³H-glycerol (5 μCi) into different pools of lipids in the presence andabsence of peptides can be determined 5 h after incubation withpeptides. Cells present in the medium and in cells can be extracted bythe method of Folch et al. (Folch, J. et al., J. Biol. Chem. 226:497-5091957). The final extracts can then be analyzed by TLC as previouslydescribed (Dashti, N. et al, J. Lipid Res. 45:1919-1928 2004).

Example 12 Effect of Peptides on Plasma Cholesterol Levels, LesionInhibition in Animal Models of Atherosclerosis, and Modulation of HDLProperties

Two mouse models can be used to study the effect of different peptideson atherosclerosis, namely apo E null mice on chow diet and LDL-R nullmice on Western diet. Apo E null mice develop atherosclerosisspontaneously on normal chow and the lesion begins to form in the aorticsinus at the age of 4 to 6 weeks. At 16 weeks of age, well definedlesions are formed at the aortic sinus. This mouse model can be used toinitiate peptide administration at 4 weeks of age and administered for 6weeks. Retroorbiral administration and administration by the tail veinrevealed a decrease in plasma cholesterol. In mouse models ofatherosclerosis (LDL-R null mice and apo E null mice) the peptides canbe administered (50 μg/mouse) retroorbitally as described above and inFIG. 2. This method enables administration of the peptide multiple timesand with minimal effect on the health of the animal. Peptides can befirst administered intravenously to apo E null and LDL-R null mice (onWestern diet) and the ability of these peptides to reduce plasmacholesterol levels can be compared. To determine the ability of thesepeptides on the fast phase of reduction of plasma cholesterol levels,plasma cholesterol levels can be measured at 2 min, 30 min, 1 h, 4 h, 8h and overnight. Using the ¹⁴C-radiolabelled peptide the kinetics ofdisappearance of the peptide from the plasma compartment can bedetermined. The organ distribution of the different peptides can also bedetermined.

The effect on lesion inhibition can be determined by performing studiesin apo E null mice on normal chow and LDL-R receptor null mice onWestern diet using the procedures described previously. 25 animals canbe used in each group. Since in LDL-R null mice the lesions develop onlywhen they are fed a Western diet, the type of lesion produced can bedifferent. Thus a careful analysis of lesion upon peptide(s)administration can provide possible differences in the mechanism bywhich these peptides inhibit atherosclerosis.

In addition lesion morphology can be selectively altered by dietarycholesterol in rabbits. Based on the literature and the resultsdescribed above, NZW rabbits fed a 1% cholesterol diet can developlesions consisting of macrophage-derived foam cells. Although earlyfoam-cell lesions in the rabbits resemble human fatty streaks, theselesions are expected to be different from the latter, forming fibrous oratheromatous plaques that are found in advanced human lesions. However,long term exposure to low levels of cholesterol in the diet has beenshown to increase the variability including advanced, fibrous plaquewhich is compensated by increase in the number of animals. To determinethe molecular events by which the peptide reduces atherosclerosis, thedifference in the macrophage content of the lesions from the control andpeptide-administered rabbits using two doses of the peptide can bedetermined. If the peptide acts directly, on the lesion formation, thesetwo doses yield different numbers of macrophage foam cells. If thepeptide acts indirectly in reducing atherosclerosis, two doses canprovide similar macrophage-foam cell numbers/lesion area. Histologicalanalysis includes stains for lipids, macrophages (using antimacrophagemonoclonal antibody Ram-1 1), and smooth muscle cells(monoclonalantibody HHF-3, directed against smooth muscle cell-specific actin todetermine SMC-rich fibrous cap formation; differences in smooth musclemigration can result with peptide administration that is related tocorrecting endothelial cell dysfunction.

It can also be determined whether the peptide inhibits atheroscleroticlesion formation by decreasing plasma cholesterol and atherogeniclipoprotein levels during the high fat diet regime compared to controlrabbits (not given the peptide). For these studies, NZW rabbits thatrespond to diet can be selected. All animals are fed a 1% cholesteroldiet and their cholesterol values determined three days after the dietinitiation. Twenty rabbits with similar cholesterol levels can beselected and changed back to a normal diet. After 15 days on a normaldiet, cholesterol values can once again be determined to check if thevalues returned to normal. Animals whose cholesterol values have notreturned to pre-diet levels can be monitored until they are normalizedor removed from the study. Ten rabbits in each group are selected andsimultaneous peptide administration and 1% diet initiation can follow.In the preliminary studies the daily average food intake and body weightwere not significantly different between control and peptideadministered group even after 6 weeks of 1% cholesterol diet and onemonth after (3 mg/kg) peptide administration (Control group food intakewas 0.16±0.02 kg, average body weight was 4.45±0.27 kg and in peptideadministered group, average food intake was 0.17±0.03 kg and averagebody weight was 4.8±0.4 kg). To study dose-response effects, smallernumber of animals (five in each group can be administered three doses ofpeptide (5 mg/kg, 3 mg/kg and 1.5 mg/kg). After the initial experiments,the dose of the peptide can be decided. A solution of the peptide(sterile saline) can be injected through the ear vein, once a week forthe duration of the study (based on previous experience, for 6 weeks).The study parameters include total cholesterol, lipoprotein levels, LOOHlevels, PON activity in the plasma. At the end of six weeks, the rabbitsare euthanized and histology assessed.

Example 13 Modulation of HDL Properties

Oxidation of LDL is associated with changes in both vascular structureand function. Activation of endothelial cells leads to an increasedexpression of adhesion molecules and chemokines such as VCAM-1, MCP-1which also enhance the accumulation of cholesterol. Under theseconditions, there is enhanced formation of reactive oxygen species(ROS), resulting in reduced levels of endothelial NO (White, C. R., etal., Proc. Natl. Acad. Sci. USA. 91:1044-1048 1994; White, C. R., etal., Proc. Natl. Acad. Sci. (USA) 93: 8745-8749 1996). It has been shownthat in WHHL rabbits Ac-hE-18A-NH₂ not only decreases atherogeniclipoprotein levels, but also remodels the existing HDL to form an apoA-I containing and peptide-containing particle that has increased PONactivity. This particle is also able to recruit lipid hydroperoxideswhich get cleared due to increased PON activity. Inhibition of LDLoxidation inhibits monocyte chemotaxis, thus, prevents its accumulationin the vessel wall and lesion formation (White, C. R., et al., Proc.Natl. Acad. Sci. (USA) 93: 8745-8749 1996). Several publications relatedto class A peptides studies have demonstrated remodeling of HDL in mouseand monkey models.

A reduction in plasma HDL is associated with impairment of reversecholesterol transport (RCT). This results in accumulation ofcell-derived cholesterol within the arterial wall, which manifests intoadvanced carotid intima-media thickening and marked susceptibility toatherosclerosis (Clee, S. M., et al. J. Clin. Invest. 106:1263-12702000). Increasing HDL in subjects with low HDL facilitates theremoval/clearance of atherogenic lipoproteins and improves endothelialfunction (Bisoendial, R. J., et al., Circulation 107:2944-2948 2003;Calabresi, L., et al., Athero. Thromb. Vasc. Biol. 23:1724-1 731 2003;Kaul, S., et al., J. Am. Coll. Cardiol. 44:1311-1319 2004). It isbelieved that peptides improve HDL function by recruiting apo A-I andPON (FIG. 19) and protects endothelial function by facilitating theremoval of atherogenic lipoproteins from the vessel wall. Removal ofLOOH increases PON activity and not PON mRNA levels or PON proteinlevels. The mechanism(s) by which this restoration takes place can bedetermined by studying the effect of the peptide on RCT using proceduresdescribed by Navab et al. (Navab, M., et al. Circulation 109:3215-3220(2004); Rader, D. J. Am. J. Cardiology. 92:42J-49J (2003)). In rabbitmodels, the amount of cholesterol excreted as bile salts and cholesterolesters can be studied using methods described by Navab et al. (Navab,M., et al., Circulation. 110: 120-125 2004). The effect of peptide(s) onABCA1-mediated cholesterol efflux in J774 macrophages can also bestudied. Macrophage ABCA1 expression can be determined by RT-PCR andWestern Blot. Macrophages are seeded in 12 well plates at a density of2×10⁶ cells/well in DMEM containing 10% FBS and allowed to attachovernight. 24 h after plating cells are labeled with ³H-cholesterol (10μCi) according to the method of Sparrow et al. After an additional 24 h,cells can be washed and media replaced with serum-free media containing0.1% BSA. Studies can be performed in the presence or absence of abromo-derivative of cAMP to determine the ABCA1-mediated cholesterolefflux and cholesterol efflux due to microsolubilization. Cholesterolefflux can be stimulated by the addition of peptide or purified A-I foran additional 24 h period. Media can then be recovered and cellssolubilized in PBS containing 0.5% Triton X-100. Radioactivity inaliquots of media and solubilized cells can then be measured.Cholesterol efflux can be analyzed by measuring radioactive counts inthe media as a percentage of total counts. It has been shown thatchlorination or nitration of Tyr in apo A-I produces dysfunctional apoA-I (Constanze, et al., Natl. Acad. Sci. U.S.A. 101:13032-13037, 2004).Radioactive tracer peptide can be used in some situations. If iodinationof peptide alters properties of the peptide, ¹⁴C-labelled peptide can beused by acetylating the peptide using ¹⁴C-acetic acid.

Measurement of paraoxanase (PON) can also be performed. The antioxidantcapacity of HDL is attributed primarily to the presence of the enzymePON. HDL isolated from mice that overexpress the gene for PON-1 ishighly resistant to LOOH formation induced by copper (Valabhji, J., etal., Clinical Science. 101:659-670 2001). A decrease in PON activity isassociated with dyslipidemia and insulin resistance in leptin- and LDLreceptor-deficient mice and diabetic humans (Valabhji, J., et al.,Clinical Science. 101:659-670 2001; Griendling, K. K. et al.,Circulation Research. 86:494-501 2000; Mertens, A., et al., Circulation.107:1640-1646 2003; Sanguinetti, S. M., et al., Diabetes, Nutrition &Metabolism-Clinical & Experimental. 14:27-36 2001; Quyyumi, A. A. Am. J.Med. 105:32S-39S 1998; Halcox, J. P., et al., Circulation. 106:653-658,2002). With this in mind, it can be determined whether chronicAc-hE-18A-NH₂, Ac-hE-4F—NH₂, and R18L-2Y administration increases PONactivity in plasma and isolated lipoprotein fractions of the two rabbitmodels. PON activity can be determined using paraoxon(O,O-diethyl-O-p-nitrophenylphosphate; Sigma Chemical Co.) as substrate.

Whether peptide administration improves endothelial function can also bedetermined. Endothelial function is compromised under conditions ofinflammation and atherogenesis (Quyyumi, A. A. Am. J. Med. 105:32S-39S1998; Halcox, J. P., et al., Circulation. 106:653-658, 2002). Defects inlipoprotein metabolism and vascular reactivity are fundamentalpathological responses to hypercholesterolemia. Extensive evidencesuggests that ROS play an important role in the initiation andprogression of these lesions (Griendling, K. K. et al., CirculationResearch. 86:494-501 2000). Blood vessels from atherosclerotic patientsand hypercholesterolemic animal models exhibit impaired,endothelium-dependent relaxation (Quyyumi, A. A. Am. J. Med. 105:32S-39S1998; Halcox, J. P., et al., Circulation. 106:653-658, 2002). NO ismodified in a hyperlipidemic environment via its reaction withsuperoxide anion (O₂), resulting in reduced NO bioactivity and yieldingthe potent oxidant peroxynitrite (ONOO) (White, C. R., et al., Proc.Natl. Acad. Sci. USA. 91:1044-1048 1994). ONOO may promote atherogenesisby reducing the beneficial physiological actions of NO and oxidizinglipoproteins (White, C. R., et al., Proc. Natl. Acad. Sci. USA.91:1044-1048 1994). Improvement in HDL function can result in a decreasein the atherogenicity of LDL which can direct LDL to a normal uptake (asapposed to scavenger receptor uptake) and thus plasma cholesterollowering. These changes are expected to increase endothelial-derived NObioactivity.

The effect of the peptides on anti-inflammatory properties can also bedetermined. Endothelial dysfunction is an early feature ofatherosclerotic disease (Quyyumi, A. A. Am. J. Med. 105:32S-39S 1998).It is an important independent clinical prognostic indicator in patientswith or without coronary artery disease. Furthermore, improvement inendothelial function is associated with improved clinical outcomes.Endothelium plays an important role in vessel homeostasis byparticipating in divergent pathophysiologic processes including vesseltone maintenance, thrombosis and inflammatory pathways associated withatherosclerosis (Halcox, J. P., et al., Circulation. 106:653-658, 2002).Endothelial dysfunction is associated with numerous factors includingdyslipidemia, hypertension, smoking and possibly genetic andenvironmental influences. Of the various pharmaceutical interventions,use of angiotensin inhibitors and statins are associated withimprovement in endothelial function. The beneficial action of statinshas been linked to lowering of total plasma cholesterol and LDL. Still,there is significant mortality and morbidity associated withatherosclerotic disease. One of the widely recognized theories ofatherosclerosis is the “response to injury” hypothesis in which oxidizedLDL causes endothelial dysfunction, leading to an insult to smoothmuscle cells and in cell proliferation. Key features of atherosclerosisare therefore the recruitment of blood monocytes to and throughendothelium, the activation/differentiation of these monocytes tomacrophages and the uptake of lipid and lipoproteins by the macrophagesto form foam cells. As such, it can be determined whether the peptidemodified HDL structure and function, leading to an improvement inendothelial function (FIG. 15). Cholesterol lowering per se cansignificantly increase NO bioavailability in isolated arteries ofhypercholesterolemic rabbits. It can also be tested whether the peptidesmodulate the binding and/or expression of pro-oxidant enzymes invascular cells.

Whether peptide administration reduces superoxide production in bloodvessels of hypercholesterolemic rabbits can also be determined. Nitricoxide becomes modified in a hyperlipidemic environment via itsinteraction with superoxide anion radical (O2), resulting in diminishedphysiological activity (White, C. R., et al., Proc. Natl. Acad. Sci.USA. 91:1044-1048 1994). Superoxide is generated in both intracellularand extracellular compartments in response to activation of pro-oxidantenzymes (NADPH oxidase, xanthine oxidase, etc) and reacts with the moremembrane-permeable and diffusible NO, yielding the potent oxidantperoxynitrite (ONOO) (White, C. R., et al., Proc. Natl. Acad. Sci. (USA)93: 8745-8749 1996; Griendling, K. K. et al., Circulation Research.86:494-501 2000). As a corollary to the studies described above, O₂production can be determined using coelenterazine-dependentchemiluminescence. The O₂-dependent oxidation of coelenterazine resultsin the formation of a high energy intermediate which emits light as itrelaxes to the ground state. A rabbit aortic segment (approximately 3 mmwide) can be placed in a vial containing 2 ml 10 μM coelenterazine-PBS.Baseline O₂ production can be monitored in tissues from peptide- orsaline-treated control animals every 30 sec for 30 min using aluminometer (BMG Labtechnologies Inc). Background chemiluminescence canbe monitored in solutions of coelenterazine-PBS in the absence ofvascular tissue. In control experiments, the specificity of thechemiluminescence signal for O₂ production can be verified by theaddition of 100 U/mL PEG-SOD and the SOD mimetictetrakis(N-ethylpyridinium-2-yl) porphyrin (T2E). These compoundslocalize to the extracellular surface and the intracellular spacerespectively and effectively scavenge O₂. The assay is calibrated bymonitoring the chemiluminescence signal of known amounts of O₂ generatedby xanthine oxidase (0.05 U) and xanthine (10 to 50 μmol/L). Rates of O₂production associated with these xanthine/xanthine oxidase incubationconditions can be determined spectrophotometically by measuring theO₂-dependent reduction of ferricytochrome C and can be normalized totissue protein.

Studies can also be performed to determine whether peptideadministration improves vascular NO release in isolated arteries ofcholesterol-fed NZW and WHHL rabbits. NO release in response to chronictreatment with peptide or saline can be assessed by monitoring theformation of the metabolites nitrate (NO₃) and nitrite (NO₂). Aorticring segments can be prepared as described above and placed in 0.5 mlPBS containing 1 μM A23187, a calcium ionophore which stimulatescellular NO formation via the calcium-dependent activation of NOS III.At the end of the 2 hr incubation period, 50 μl samples of PBS can becollected. Nitrate in this sample can be enzymatically reduced to NO₂ bytreatment with E. coli enriched nitrate reductase. Total NO₂ can be usedas an index of NO production (Zhang, C., et al. J. Biol. Chem. 276:27159-27165 2001). Nitrite can be detected in the nM range using thefluorophore 2,3-diaminonaphthalene (DAN) (Calbiochem, Inc.). Underalkaline conditions, DAN converts NO2 to the fluorescent compound1(H)-naphthotriazole. Nitrite concentration can then be monitored by thespectrofluorometric excitation of 1(H)-naphthotriazole (360 nm andemission at 450 nm). A standard curve can be constructed for NaNO₂(1-1,000 nM) in order to convertfluorescence intensity values toconcentrations. Nitrite formation will be normalized to proteinconcentration. In additional experiments, plasma levels of NOmetabolites isolated from peptide- and saline-treated experimentalanimals can be measure.

Further experiments can be performed to determine whether theadministration of the peptides influences the expression/activity ofpro-oxidant enzymes in arteries of cholesterol-fed NZW and WHHL rabbits.Previous studies showed that an increase in plasma cholesterol incholesterol-fed NZW rabbits was associated with the release of thepro-oxidant enzyme xanthine oxidase (XO) into the circulation and itsconcentration at HSPG binding sites on the vascular endothelium (White,C. R., et al., Proc. Natl. Acad. Sci. (USA) 93: 8745-8749 1996; Adachi,T., et al., Biochemical J. 289(2):523-527 1993). At this site, XO servedas a source of O2 and contributed to the development of endothelialdysfunction. The inhibition of relaxation associated with XO bindingcould be reversed by addition of heparin, allopurinol, and chimericheparin-binding superoxide dismutase. The identification of XO invascular lesions of humans suggests that the enzyme can be a clinicallyrelevant target for the therapeutic treatment of atherosclerosis (Swain,J. et al. FEBS Lett. 368(3):513-515 1995). This is underscored byfindings that infusion of the XO inhibitor oxypurinol in humansincreases forearm blood flow in HC, but not hypertensive, patients(Cardillo, C., et al., Hypertension 30:57-63 1997).

It has been reported previously that chronic elevation of plasmacholesterol in rabbits induces an increase in circulating xanthineoxidase concentration. The liver and intestine are principal sources ofcirculating XO. Cholesterol accumulation in the liver is associated withhepatocellular injury and increased conversion of xanthine dehydrogenase(XDH) to xanthine oxidase (XO). Increased plasma levels of alaninetransaminase (ALT) are additionally associated with XO release in thecirculation. Circulating XO readily binds to endothelial cell surfaceheparan sulfate proteoglycans (HSPG) and becomes endocytosed, thusinducing oxidative injury in both extracellular and intracellularcompartments at distal sites. The administration of the peptide(s) canexert vascular protective effects via two mechanisms. First, as shownabove, peptide-administration effectively reduces total plasmacholesterol which is predicted to reduce cholesterol-induced hepaticinjury in hypercholesterolemic rabbits and circulating plasma XOactivity. In addition, the peptides, due to their ability to interactwith HSPG, can compete with and displace XO from the same cell surfacebinding sites. This action can reduce XO-mediated oxidant injury to theendothelium and underlying VSMCs. Under these conditions, circulating XOactivity can be increased, but the chronic peptide(s) treatment canprevent XO binding to endothelial cells and reduces the formation of ROSat this site. Xanthine oxidase activity of plasma and tissues fromcontrol and hypercholesterolemic rabbits can be measured using HPLC(White, C. R., et al., Proc. Natl. Acad. Sci. (USA) 93: 8745-8749 1996).At sacrifice, plasma samples are obtained from test animals andimmediately frozen at −80° C. Prior to measuring enzymatic activity,endogenous urate can then be removed by passing the sample over aSephadex G-25 column. Samples can then be treated with oxonic acid (2mM) to inhibit plasma uricase activity. Xanthine (75 μM) can be added,and XO activity assessed by monitoring the production of urate. Thesereactions are performed in the absence and presence of NAD (0.5 mM) andpyruvic acid (5 mM) in order to assess XO and total oxidoreductase(XO+XDH) activity, respectively. The specificity of this detectionmethod for urate production by XO/XDH can be verified by inhibition ofurate formation following allopurinol addition in some samples. XOprotein content of homogenized arteries can be assessed by Western blotusing a commercially available monoclonal anti-XO antibody (UnitedStates Biologicals). Effects of peptides treatment on XObinding/localization to the vascular wall can also be tested byimmunohistochemistry using a commercially available XO antibody.

Alternatively, peptides can target the expression of NADPH oxidase, anadditional source of vascular superoxide in arteries ofhypercholesterolemic animals. Accordingly, real time polymerase chainreaction (RT-PCR) can be used to quantitate mRNA for p22^(phox)acritical subunit of the NADPH oxidase, in aortic tissues ofcholesterol-fed NZW and WHHL rabbits. Total RNA can be extracted fromaortas using TRIzol Reagent, and p22^(phox)mRNA analyzed by RT-PCR.p22^(phox)mRNA can be co-amplified with GAPDH mRNA in a Techne ThermalCycler PHC-3. PCR products can be analyzed on a 1.2% agarose-ethidiumbromide gel. The gels can then be photographed, and the intensity of theindividual p22^(phox) and GAPDH mRNA bands measured by laserdensitometric scanning, using a Molecular Dynamics PersonalDensitometer. Changes in p22^(phox)mRNA levels are expressed as arelative ratio of mRNA band intensity to that of GAPDH.

The effects of peptide administration on functional responses ofarteries in two rabbit models can also be performed. Cholesterol-feedingof rabbits has been widely used to study the effects ofhypercholesterolemia on vascular function and lipid oxidation (White, C.R., et al., Proc. Natl. Acad. Sci. USA. 91:1044-1048, 1994; Geetanjali,B., et al., Cardiovascular Pathology 11: 97-103 2002). Previous studieshave shown a significant increase in plasma cholesterol levels inhypercholesterolemic NZW rabbits that are characterized by an increasein βVLDL content. The WHHL rabbit is also commonly used to studymechanisms of atherogenesis. WHHL rabbits are also hyperlipidemic, and,in contrast to cholesterol-fed NZW rabbits, exhibit increased plasmalevels of LDL cholesterol. Experiments can be performed to assess theeffects of peptide administration on endothelium-dependent relaxantresponses in arteries of cholesterol-fed NZW and WHHL rabbits.

For these experiments, many animals described above can be used. NewZealand white rabbits (2.5-3.0 kg) (Myrtle Farms, Inc.) can be fedmodified laboratory chow (Purina, Inc.) containing 1% cholesterol for 6weeks. WHHL rabbits (Covance Inc.) plasma cholesterol levels areapproximately 80 mg/dl and increase to 600±200 mg/dl by 6 months.Rabbits from each group are assigned at random to receive eitherpeptides or saline (administered by i.v. infusion via the marginal earartery) at 3 mg/kg/week for 7 to 8 weeks. After the treatment period,rabbits are euthanized, and the aorta can be excised and cleansed of fatand adhering tissue. Isometric tension can be measured as describedpreviously (White, C. R., et al., Proc. Natl. Acad. Sci. USA.91:1044-1048 1994; White, C. R., et al., Proc. Natl. Acad. Sci. (USA)93: 8745-8749 1996). The vessel can then be cut into individual ringsegments (3-4 mm in width) and suspended from a force-displacementtransducer in a tissue bath. Ring segments can be bathed in abicarbonate-buffered, Krebs-Henseleit (K-H) solution of the followingcomposition (mM): NaCl 118; KCl 4.6; NaHCO₃ 27.2; KH₂PO₄ 1.2; MgSO₄ 1.2;CaCl₂1.75; Na₂EDTA 0.03, and glucose 11.1. A passive load of 3 g can beapplied to all ring segments and maintained at this level throughout theexperiment. At the beginning of each experiment, indomethacin-treatedring segments can be depolarized with KCl (70 mM) to determine themaximal contractile capacity of the vessel. Indomethacin is added underthese conditions to inhibit the formation of cycloxygenase-derivedvasoactive metabolites. Rings can then be thoroughly washed and allowedto equilibrate. In subsequent experiments, vessels can be submaximallycontracted (40% of KCl response) with PE (3×10⁸10⁷M). When tensiondevelopment reaches a plateau, acetylcholine (Ach: 10⁹ to 3×10⁶M) can beadded cumulatively to the bath to invoke endothelium-dependentrelaxation. At the end of each dose response protocol, sodiumnitroprusside (SNP: 5 μM) can be added to elicit residualendothelium-independent relaxation. Real time data can be collected forall experiments and downloaded to an IBM PC for analysis usingcommercially available software. Preliminary data indicates thatpeptide-treated animals show restored endothelial function. Thesestudies establish if this is so in both diet-induced and genetic modelsof atherosclerosis.

Morphometric analysis of aortic tissues can be subsequently performed todetermine the effect of peptide treatment on fatty streak lesionformation. Lesion areas can be assessed using light microscopy andoil-red-O staining (Navab, M., et al., J. Lipid Res. 41:1495-1508 2000;Garber, D. W. et al., J. Lipid Res. 42:545-552 2001). Cholesterolcontent of the artery wall can also be performed using techniquesdescribed by Thorngate et al (Throngate, F. E. et al., Arterio. Thromb.Vasc. Biol. 20:1939-1945 2000).

Example 14

The effects of some of the disclosed apolipoprotein E-mimicking peptideson plasma cholesterol were investigated. ZDFfa/fa (Zucker diabetic fattyrats with a defect in their leptin receptor) male rats (5-6 weeks,180-220 g) were obtained from Charles River Laboratories Inc. The ratswere housed in individual cages and allowed to acclimatize for a fewdays prior to performing any intervention. The rats' diet consisted of a2016 Teklad Global 16% Protein Rodent Diet. Close monitoring of thedietary intake was performed. Water was provided ad-libitum.

Rats were then divided into various groups (n=7-8/group): control(saline) and peptide. Animals were fasted overnight (12 h) prior toblood draws. Animals were individually administered one of the peptides(5 mg/Kg) or saline via the tail vein. (See FIG. 20 for a diagram of thetimeline of administration). Any weight changes were monitored closely.Blood was obtained at baseline, and at pre-specified intervals. Plasmawas separated and aliquoted.

Analysis on the blood extracted consisted of a cholesterol and anendocrine assay. The cholesterol assay was carried out using acolorimetric cholesterol assay. The assay was performed using aCholesterol reagent (ThermoDMA, Arlington, Tex.). The endocrine assaywas performed by Millipore, Inc using a Lincoplex multi-analyte ratendocrine kit. Rat adipnectin was measured by Millipore, Inc using mouseadiponectin RIA methodology.

Results

As described above, peptides Ac-hE-18A-NH₂, Ac-hE-4F—NH₂ and Ac-hE-Sc18Awere administered (iv) to apo E null mice (n=4) and plasma cholesterolvalues were determined at before administration (0 min), 5 min and 2 hafter administration. Results are shown in FIG. 22. While Ac-hE-18A-NH₂and Ac-hE-4F—NH₂ showed a large reduction in plasma cholesterol levelsat 2 h time point, peptide Ac-hE-Sc18A-NH₂ did not show such as great ofa difference, however, plasma cholesterol levels were decreased.

1. A method of treating cancer in a subject, comprising administering aneffective amount of a synthetic apolipoprotein E-mimicking peptide tothe subject.
 2. The method of claim 1, wherein the syntheticapolipoprotein E-mimicking peptide comprises a sequence selected fromthe group consisting of SEQ ID NOs: 11-14, 18-57, 60, 61, and 62-103. 3.The method of claim 1, wherein the synthetic apolipoprotein E-mimickingpeptide comprises the sequence of SEQ ID NO: 62 or SEQ ID NO:
 12. 4. Themethod of claim 1, wherein the synthetic apolipoprotein E-mimickingpeptide comprises a receptor binding domain peptide and alipid-associating peptide, wherein the lipid-associating peptide iscovalently linked to the receptor binding domain peptide.
 5. The methodof claim 4, wherein the receptor binding domain peptide is from aspecies selected from the group consisting of human, mouse, rabbit,monkey, rat, bovine, pig and dog.
 6. The method of claim 4, wherein thereceptor binding domain peptide comprises a sequence selected from thegroup consisting of SEQ ID NOs: 1-2, 3, 5-10, 15, and
 58. 7. The methodof claim 4, wherein the receptor binding domain peptide is mutated 8.The method of claim 4, wherein the receptor binding domain peptide isscrambled.
 9. The method of claim 4, wherein the receptor binding domainpeptide is in a reversed orientation.
 10. The method of claim 4, whereinthe lipid-associating peptide is model class A amphipathic helicalpeptide 18A.
 11. The method of claim 4, wherein the lipid-associatingpeptide comprises a sequence selected from the group consisting of SEQID NOs: 4, 16, 17, and
 59. 12. The method of claim 4, wherein thelipid-associating peptide is mutated.
 13. The method of claim 4, whereinthe lipid-associating peptide is scrambled.
 14. The method of claim 4,wherein the lipid-associating peptide is in a reversed orientation. 15.The method of claim 4, wherein the receptor binding domain is covalentlylinked to the lipid-associating peptide in a domain switchedorientation.
 16. The method of claim 1, wherein the syntheticapolipoprotein E-mimicking peptide is protected using acetyl and amidegroups at the N- and C-terminus, respectively.
 17. The method of claim1, wherein the synthetic apolipoprotein E-mimicking peptide isadministered in a composition comprising a pharmaceutically acceptablecarrier.
 18. A method of treating cancer in a subject, comprisingadministering an effective amount of a pharmaceutical compositioncomprising a synthetic apolipoprotein E-mimicking peptide and apharmaceutically acceptable carrier to the subject.
 19. The method ofclaim 18, wherein the synthetic apolipoprotein E-mimicking peptidecomprises a sequence selected from the group consisting of SEQ ID NOs:11-14, 18-57, 60, 61, and 62-103.
 20. The method of claim 18, whereinthe synthetic apolipoprotein E-mimicking peptide comprises the sequenceof SEQ ID NO: 62 or SEQ ID NO:
 12. 21. The method of claim 18, whereinthe synthetic apolipoprotein E-mimicking peptide comprises a receptorbinding domain peptide and a lipid-associating peptide, wherein thelipid-associating peptide is covalently linked to the receptor bindingdomain peptide.
 22. The method of claim 21, wherein the receptor bindingdomain peptide is from a species selected from the group consisting ofhuman, mouse, rabbit, monkey, rat, bovine, pig and dog.
 23. The methodof claim 21, wherein the receptor binding domain peptide comprises asequence selected from the group consisting of SEQ ID NOs: 1-2, 3, 5-10,15, and
 58. 24. The method of claim 21, wherein the receptor bindingdomain peptide is mutated
 25. The method of claim 21, wherein thereceptor binding domain peptide is scrambled.
 26. The method of claim21, wherein the receptor binding domain peptide is in a reversedorientation.
 27. The method of claim 21, wherein the lipid-associatingpeptide is model class A amphipathic helical peptide 18A.
 28. The methodof claim 21, wherein the lipid-associating peptide comprises a sequenceselected from the group consisting of SEQ ID NOs: 4, 16, 17, and
 59. 29.The method of claim 21, wherein the lipid-associating peptide ismutated.
 30. The method of claim 21, wherein the lipid-associatingpeptide is scrambled.
 31. The method of claim 21, wherein thelipid-associating peptide is in a reversed orientation.
 32. The methodof claim 21, wherein the receptor binding domain is covalently linked tothe lipid-associating peptide in a domain switched orientation.
 33. Themethod of claim 18, wherein the synthetic apolipoprotein E-mimickingpeptide is protected using acetyl and amide groups at the N- andC-terminus, respectively.
 34. A method of treating a subject with cancercomprising: selecting a subject with cancer; administering an effectiveamount of a synthetic apolipoprotein E-mimicking peptide to the subject,thereby treating cancer in the subject.
 35. The method of claim 34,wherein the synthetic apolipoprotein E-mimicking peptide comprises asequence selected from the group consisting of SEQ ID NOs: 11-14, 18-57,60, 61, and 62-103.
 36. The method of claim 34, wherein the syntheticapolipoprotein E-mimicking peptide comprises the sequence of SEQ ID NO:62 or SEQ ID NO:
 12. 37. The method of claim 34, wherein the syntheticapolipoprotein E-mimicking peptide comprises a receptor binding domainpeptide and a lipid-associating peptide, wherein the lipid-associatingpeptide is covalently linked to the receptor binding domain peptide. 38.The method of claim 37, wherein the receptor binding domain peptide isfrom a species selected from the group consisting of human, mouse,rabbit, monkey, rat, bovine, pig and dog.
 39. The method of claim 37,wherein the receptor binding domain peptide comprises a sequenceselected from the group consisting of SEQ ID NOs: 1-2, 3, 5-10, 15, and58.
 40. The method of claim 37, wherein the receptor binding domainpeptide is mutated
 41. The method of claim 37, wherein the receptorbinding domain peptide is scrambled.
 42. The method of claim 37, whereinthe receptor binding domain peptide is in a reversed orientation. 43.The method of claim 37, wherein the lipid-associating peptide is modelclass A amphipathic helical peptide 18A.
 44. The method of claim 37,wherein the lipid-associating peptide comprises a sequence selected fromthe group consisting of SEQ ID NOs: 4, 16, 17, and
 59. 45. The method ofclaim 37, wherein the lipid-associating peptide is mutated.
 46. Themethod of claim 37, wherein the lipid-associating peptide is scrambled.47. The method of claim 37, wherein the lipid-associating peptide is ina reversed orientation.
 48. The method of claim 37, wherein the receptorbinding domain is covalently linked to the lipid-associating peptide ina domain switched orientation.
 49. The method of claim 34, wherein thesynthetic apolipoprotein E-mimicking peptide is protected using acetyland amide groups at the N- and C-terminus, respectively.
 50. The methodof claim 34, wherein the synthetic apolipoprotein E-mimicking peptide isadministered in a composition comprising a pharmaceutically acceptablecarrier.
 51. A method of treating cancer in a subject, comprising:selecting a subject with cancer; and administering an effective amountof a pharmaceutical composition comprising a synthetic apolipoproteinE-mimicking peptide and a pharmaceutically acceptable carrier to thesubject, thereby treating cancer in the subject.
 52. The method of claim51, wherein the synthetic apolipoprotein E-mimicking peptide comprises asequence selected from the group consisting of SEQ ID NOs: 11-14, 18-57,60, 61, and 62-103.
 53. The method of claim 51, wherein the syntheticapolipoprotein E-mimicking peptide comprises the sequence of SEQ ID NO:62 or SEQ ID NO:
 12. 54. The method of claim 51, wherein the syntheticapolipoprotein E-mimicking peptide comprises a receptor binding domainpeptide and a lipid-associating peptide, wherein the lipid-associatingpeptide is covalently linked to the receptor binding domain peptide. 55.The method of claim 51, wherein the receptor binding domain peptide isfrom a species selected from the group consisting of human, mouse,rabbit, monkey, rat, bovine, pig and dog.
 56. The method of claim 54,wherein the receptor binding domain peptide comprises a sequenceselected from the group consisting of SEQ ID NOs: 1-2, 3, 5-10, 15, and58.
 57. The method of claim 54, wherein the receptor binding domainpeptide is mutated
 58. The method of claim 54, wherein the receptorbinding domain peptide is scrambled.
 59. The method of claim 54, whereinthe receptor binding domain peptide is in a reversed orientation. 60.The method of claim 54, wherein the lipid-associating peptide is modelclass A amphipathic helical peptide 18A.
 61. The method of claim 54,wherein the lipid-associating peptide comprises a sequence selected fromthe group consisting of SEQ ID NOs: 4, 16, 17, and
 59. 62. The method ofclaim 54, wherein the lipid-associating peptide is mutated.
 63. Themethod of claim 54, wherein the lipid-associating peptide is scrambled.64. The method of claim 54, wherein the lipid-associating peptide is ina reversed orientation.
 65. The method of claim 54, wherein the receptorbinding domain is covalently linked to the lipid-associating peptide ina domain switched orientation.
 66. The method of claim 51, wherein thesynthetic apolipoprotein E-mimicking peptide is protected using acetyland amide groups at the N- and C-terminus, respectively.